Methods and compositions for controlling pests

ABSTRACT

Pest-controlling compositions include one or more β-diones, particularly β-diketones and β-triketones, and are used inter alia for preventing, eradicating, destroying, repelling or mitigating harmful, annoying or undesired pests including insects, arachnids, helminths, molluscs, protozoa and viruses. β-diones can be prepared by de novo synthesis or from natural sources such as volatile oil-bearing plants from families including Alliaceae, Apiaceae, Asteraceae, Cannabinaceae, Lamiaceae, Pteridaceae, Myrtaceae, Myoporaceae, Proteaceae, Rutaceae and Zingiberaceae.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/250,187, filed Apr. 10, 2014, which is a continuation of U.S.application Ser. No. 13/431,726, filed Mar. 27, 2012, which is acontinuation of U.S. application Ser. No. 12/782,125, filed May 18,2010, which is a continuation of U.S. application Ser. No. 12/134,035,filed Jun. 5, 2008, and granted Oct. 26, 2010 as U.S. Pat. No.7,820,209, which is a continuation of U.S. application Ser. No.10/477,057, filed Sep. 23, 2004. U.S. application Ser. No. 10/477,057 isthe U.S. National Phase under 35 U.S.C. §371 of InternationalApplication No. PCT/AU02/00569, filed May 8, 2002 designating the U.S.and published in English as WO 02/089587, which claims priority toAustralian Patent Application PR 4842, filed May 8, 2001. Thisapplication incorporates herein by reference U.S. application Ser. Nos.13/431,726, 12/782,125, 12/134,035, and 10/477,057, U.S. Pat. No.7,820,209, International Application No. PCT/AU02/00569 including theInternational Publication No. WO 02/089587, and Australian PatentApplication PR 4842 in their entireties.

FIELD OF THE INVENTION

This invention relates generally to methods and compositions forcontrolling pests. More particularly, the present invention relates topest-controlling compositions comprising as active ingredients one ormore β-diones, particularly β-diketones and β-triketones, and to the useof these compositions inter alia for preventing, eradicating,destroying, repelling or mitigating harmful, annoying or undesired pestsincluding insects, arachnids, helminths, molluscs, protozoa and viruses.The present invention further relates to processes of preparing β-dionesby de novo synthesis or from natural sources such as volatileoil-bearing plants from families including Alliaceae, Apiaceae,Asteraceae, Cannabinaceae, Lamiaceae, Pteridaceae, Myrtaceae,Myoporaceae, Proteaceae, Rutaceae and Zingiberaceae. Bibliographicdetails of various publications referred to in this specification arecollected at the end of the description.

BACKGROUND OF THE INVENTION

Triketones have been used for many years as herbicides for the controlof undesired vegetation. Herbicidal triketones have been described, forexample, in EP-A-338992, EP-A-336898, U.S. Pat. No. 4,869,748,EP-A-186118, EP-A-186119, EP-A-186120, U.S. Pat. No. 4,202,840, U.S.Pat. No. 4,695,673, U.S. Pat. No. 4,780,127, U.S. Pat. No. 4,921,526,U.S. Pat. No. 5,006,150, U.S. Pat. No. 5,545,607, U.S. Pat. No.5,925,795, U.S. Pat. No. 5,990,046, U.S. Pat. No. 6,218,579,EP-A-249150, EP-A-137963, EP-A-394889, EP-A-506907 or EP-B-135191.Examples of herbicidal triketones are inter alia Sulcotrione (MIKADO®)whose chemical designation is2-(2-chloro-4-methanesulfonylbenzoyl)-1,3-cyclohexandione,2-(4-methylsulfonyloxy-2-nitrobenzoyl)-4,4,6,6-tetramethyl-1,3-cyclohexanedione;3-(4-methylsulfonyloxy-2-nitrobenzoyl)-bicyclo-[3,2,1]octane-2,4-dione;3-(4-methylsulfonyl-2-nitrobenzoyl)-bicyclo-[3,2,1]octane-2,4-dione;4-(4-chloro-2-nitrobenzoyl)-2,6,6-trimethyl-2H-1,2-oxazine-3,5(4H,6H)-dione;3-(4-methylthio-2-nitrobenzoyl)-bicyclo[3,2,1]octane-2,4-dione;4-(2-nitro-4-trifluoromethoxybenzoyl)-2,6,6-trimethyl-2H-1,2-oxazine-3,5(4H,6H)-dione.

SUMMARY OF THE INVENTION

The instant invention is predicated in part on the discovery thatβ-diones, particularly β-diketones and β-triketones, such as thoseobtainable from volatile oil-bearing plants including plants from thefamilies Alliaceae, Apiaceae, Asteraceae, Cannabinaceae, Lamiaceae,Pteridaceae, Myrtaceae, Myoporaceae, Proteaceae, Rutaceae andZingiberaceae, exhibit significant pesticidal, especially insecticidal,arachnicidal, helminthicidal and/or molluscicidal activity. Thisdiscovery has been reduced to practice in novel pest-controllingcompositions and methods for their preparation and use, as describedhereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structures relating to the major constituents of thepublished Myrtaceae essential oils.

FIG. 2 is a representation of a GC-MS trace of E. cloeziana oil.

FIG. 3 is a tabular and graphical representation showing ¹H NMR datarecorded on a fraction (F4) obtained from silica gel chromatography ofE. cloeziana oil and the structure of the major and minor isomers of thecompound deduced from these data.

FIG. 4 is a diagrammatic representation showing various tautomeric formsof an isolated β-triketone compound in solution (CDCl₃).

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention contemplates the use of a β-dionecompound, particularly a β-diketone or a β-triketone compound, in thepreparation of a composition for controlling harmful, annoying orundesired pests, said compound being represented by the general formula(I)

wherein

A is (C═O)R₁, (C═S)R₁, OR₂, SR₂, (CR₃NR₄R₅), C(R₃)₂OR₂, NR₄R₅,(C═N—R₄)R₁, N═O, N(═O)₂, NR₄OR₂ or SO₄R₂;

R₁ is selected from H, C₁-C₁₀ alkyl, C₂-C₁₀ arylalkyl, C₃-C₆ cycloalkyl,C₂-C₁₀ alkenyl, C₂-C₁₀ heteroarylalkyl, C₁-C₁₀ haloalkyl, C₁-C₁₀dihaloalkyl, C₂-C₁₀ trihaloalkyl, C₂-C₁₀ haloalkoxy, C₁-C₁₀hydroxyalkyl, C₁-C₁₀ thioalkyl and C₁-C₁₀ nitroalkyl, OR₂, SR₂,(CR₃NR₄R₅), NR₄R₅, (C═N—R₄)R₆, N═O, N(═O)₂, NR₄OR₇ or SO₄R₇;

R₂ is selected from H, C₁-C₁₀ alkyl, C₂-C₁₀ arylalkyl, C₃-C₆ cycloalkyl,C₂-C₁₀ alkenyl, C₂-C₁₀ heteroarylalkyl, C₂-C₁₀ haloalkyl, C₂-C₁₀dihaloalkyl, C₂-C₁₀ trihaloalkyl, (CR₃NR₄R₅), NR₄R₅, (C═N—R₄)R₆, N═O,N(═O)₂ or NR₄OR₇;

R₃ is selected from H, C₁-C₁₀ alkyl, C₂-C₁₀ arylalkyl, C₃-C₆ cycloalkyl,C₂-C₁₀ alkenyl, C₂-C₁₀ heteroarylalkyl, C₂-C₁₀ haloalkyl, C₂-C₁₀dihaloalkyl, C₂-C₁₀ trihaloalkyl, C₂-C₁₀ haloalkoxy, OR₇, SR₇,(CR₈NR₄R₅), NR₄R₅, (C═N—R₄)R₆, N═O, N(═O)₂, NR₄OR₇ or SO₄R₇;

R₄ and R₅ are independently selected from H, C₁-C₁₀ alkyl, C₂-C₁₀arylalkyl, C₃-C₆ cycloalkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ heteroarylalkyl,C₂-C₁₀ haloalkyl, C₂-C₁₀ dihaloalkyl, C₂-C₁₀ trihaloalkyl, OR₇ or SR₇;

R₆ is selected from H, C₁-C₁₀ alkyl, C₂-C₁₀ arylalkyl, C₃-C₆ cycloalkyl,C₂-C₁₀ alkenyl, C₂-C₁₀ heteroarylalkyl, C₂-C₁₀ haloalkyl, C₂-C₁₀dihaloalkyl, C₂-C₁₀ trihaloalkyl, C₂-C₁₀ haloalkoxy, OR₇, SR₇,(CR₈NR₉R₁₀), NR₉R₁₀ or NR₉OR₇; R₇ is selected from H, C₁-C₁₀ alkyl,C₂-C₁₀ arylalkyl, C₃-C₆ cycloalkyl, C₂-C₁₀ alkenyl, C₂-C₁₀heteroarylalkyl, C₂-C₁₀ haloalkyl, C₂-C₁₀ dihaloalkyl, C₂-C₁₀trihaloalkyl;

R₈ is selected from H, C₁-C₁₀ alkyl, C₂-C₁₀ arylalkyl, C₃-C₆ cycloalkyl,C₂-C₁₀ alkenyl, C₂-C₁₀ heteroarylalkyl, C₂-C₁₀ haloalkyl, C₂-C₁₀dihaloalkyl, C₂-C₁₀ trihaloalkyl, OR₁₁, SR₁₁ or NR₉R₁₀;

R₉ and R₁₀ are independently selected from H, C₁-C₁₀ alkyl, C₂-C₁₀arylalkyl, C₃-C₆ cycloalkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ heteroarylalkyl,C₂-C₁₀ haloalkyl, C₂-C₁₀ dihaloalkyl, C₂-C₁₀ trihaloalkyl, OR₁₂ or SR₁₂;

R₁₁ is selected from H, C₁-C₁₀ alkyl, C₂-C₁₀ arylalkyl, C₃-C₆cycloalkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ heteroarylalkyl, C₂-C₁₀ haloalkyl,C₂-C₁₀ dihaloalkyl, C₂-C₁₀ trihaloalkyl;

R₁₂ is selected from H, C₁-C₁₀ alkyl, C₂-C₁₀ arylalkyl, C₃-C₆cycloalkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ heteroarylalkyl, C₂-C₁₀ haloalkyl,C₂-C₁₀ dihaloalkyl, C₂-C₁₀ trihaloalkyl;

B is H, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, aryl or heteroaryl;

X and Y are independently selected from oxygen, sulfur, —N—R₄; and

Q completes a 5-8-member saturated or unsaturated carbocyclic orheterocyclic ring in which optionally one or more members comprise—C(═X)—; and wherein Q is optionally substituted with one or moresubstituents selected from C₁-C₁₀ alkyl, C₃-C₆ cycloalkyl, C₂-C₁₀alkenyl, C₂-C₁₀ haloalkyl, C₂-C₁₀ dihaloalkyl, C₂-C₁₀ trihaloalkyl,C₂-C₁₀ haloalkoxy, OR₂, SR₂, (CR₃NR₄R₅), NR₄R₅, (C═N—R₄)R₁, N═O, N(═O)₂,NR₄OR₂, SO₄R₂, C₂-C₁₀ 1-arylalkyl, C₂-C₁₀ 2-arylalkyl or (C═X)R₁.

Heterocyclic systems can be optionally attached to a moiety other thanthose set forth above via a carbon atom or a heteroatom of R₁ to R₁₁.

Preferred compounds represented by formula (I) are β-diketones andespecially preferred are β-triketones.

As used herein, the term “alkyl” refers to linear or branched chains.The term “haloalkyl” refers to an alkyl group substituted by at leastone halogen. Similarly the term “haloalkoxy” refers to an alkoxy groupsubstituted by at least one halogen. As used herein the term “halogen”refers to fluorine, chlorine, bromine and iodine.

As used herein the term “aryl” refers to aromatic carbocyclic ringsystems such as phenyl or naphthyl, anthracenyl, especially phenyl.Suitably, aryl is C₆-C₁₄ with mono, di, tri, tetra and pentasubstitution containing OR₂, F, Cl, Br, I, NO₂, CF₃, COR₁, NR₄R₅, SO₂R₂,SR₂.

As used herein the terms “heterocycle”, “heterocyclic”, “heterocyclicsystems” and the like refer to a saturated, unsaturated, or aromaticcarbocyclic group having a single ring, multiple fused rings (forexample, bicyclic, tricyclic, or other similar bridged ring systems orsubstituents), or multiple condensed rings, and having at least oneheteroatom such as nitrogen, oxygen, or sulfur within at least one ofthe rings. This term also includes “heteroaryl” which refers to aheterocycle in which at least one ring is aromatic. Any heterocyclic orheteroaryl group can be unsubstituted or optionally substituted with oneor more groups, as defined above. Further, bi- or tricyclic heteroarylmoieties may comprise at least one ring, which is either completely, orpartially, saturated. Suitable heteroaryl moieties include, but are notlimited to oxazolyl, thiazaoyl, thienyl, furyl, 1-isobenzofuranyl,2H-pyrrolyl, N-pyrrolyl, imidazolyl, pyrazolyl, isothiazolyl,isooxazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyradazinyl, indolizinyl,isoindolyl, indoyl, indolyl, purinyl, phthalazinyl.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

A preferred carbocyclic ring formed by Q is an optionally substitutedcyclohexanedione.

A preferred subgroup of compounds of formula (I) is represented byformula (II)

Such compounds may exist in a number of tautomeric forms. For example,in the case wherein X and Y are each oxygen, and B is hydrogen, then thecompounds of formula II may exist as one or more of the structuralformulae shown below.

It is intended that all such tautomeric structures are included withinthe scope of the present invention.

It should also be appreciated that some of the compounds of formula (I)are capable of existing as different geometric isomers anddiastereomers. The invention thus includes both the individual isomersand mixtures of such isomers.

Another preferred subgroup of compounds of formula (I) is represented byformula (III)

wherein

X, Y and Z are each independently selected from oxygen, sulfur, —N—R₄ orone of C═X, C═Y or C═Z is CH₂;

A is (C═O)R₁, (C═S)R₁, OR₂, SR₂, (CR₃NR₄R₅), C(R₃)₂OR₂, NR₄R₅,(C═N—R₄)R₁, N═O, N(═O)₂, NR₄OR₂ or SO₄R₂;

B is as defined above;

C, D, E and F are each independently selected from H, C₁-C₁₀ alkyl,C₃-C₆ cycloalkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ haloalkyl, C₂-C₁₀ dihaloalkyl,C₂-C₁₀ trihaloalkyl, OR₂, SR₂, (CR₃NR₄R₅), NR₄R₅, (C═N—R₄)R₁, N═O,N(═O)₂, NR₄OR₂, SO₄R₂, C₂-C₁₀ 1-arylalkyl, C₂-C₁₀ 2-arylalkyl or(C═X)R₁; and

R₁, R₂, R₂, R₄ and R₅ are as defined above.

Preferred β-diones represented by formula (III) are flavesone(1-isobutyroyl-3,3,5,5-tetramethylcyclohexan-2,4,6-trione),isoleptospermone(1-isovaleroyl-3,3,5,5-tetramethylcyclohexan-2,4,6-trione),leptospermone (1-valeroyl-3,3,5,5-tetramethylcyclohexan-2,4,6-trione),papuanone (1-pentoyl-3,3,5,5-tetramethylcyclohexan-2,4,6-trione),grandiflorone(1-(2-phenylethyl)-3,3,5,5-tetramethylcyclohexan-2,4,6-trione) andjensenone (1-valeroyl-3,5-dicarbonylcyclohexan-2,4,6-trione), includinganalogues and derivatives thereof.

By way of example, flavesone analogues contemplated by the presentinvention include, but are not restricted to, compounds having thefollowing structural formulae, wherein the structural formula offlavesone is shown for comparative purposes:

Non-limiting examples of isoleptospermone analogues contemplated by thepresent invention include, but are not restricted to, compounds havingthe following structural formulae, wherein the structural formula ofisoleptospermone is shown for comparative purposes:

Non-limiting examples of leptospermone analogues contemplated by thepresent invention include, but are not restricted to, compounds havingthe following structural formulae, wherein the structural formula ofleptospermone is shown for comparative purposes:

Non-limiting examples of jensenone analogues contemplated by the presentinvention include, but are not restricted to, compounds having thefollowing structural formulae, wherein the structural formula ofjensenone is shown for comparative purposes:

Another preferred subgroup of compounds of formula (I) is represented byformula (IV)

wherein

X and Y are each independently selected from oxygen, sulfur —N—R₄ or oneof C═X or C═Y is CH₂;

A is (C═O)R₁, (C═S)R₁, OR₂, SR₂, (CR₃NR₄R₅), C(R₃)₂OR₂, NR₄R₅,(C═N—R₄)R₁, N═O, N(═O)₂, NR₄OR₂ or SO₄R₂;

-   -   B is as defined above;    -   C, D, E and F are each independently selected from H, C₁-C₁₀        alkyl, C₂-C₁₀ arylalkyl, C₃-C₆ cycloalkyl, C₂-C₁₀ alkenyl,        C₂-C₁₀ heteroarylalkyl, C₂-C₁₀ haloalkyl, C₂-C₁₀ dihaloalkyl,        C₂-C₁₀ trihaloalkyl, C₂-C₁₀ haloalkoxy, OR₂, SR₂, (CR₃NR₄R₅),        NR₄R₅, (C═N—R₄)R₁, N═O, N(═O)₂, NR₄OR₂, SO₄R₂; and R₁, R₂, R₂,        R₄ and R₅ are as defined above.

Preferred β-diones represented by formula (IV) are tasmanone(1-isobutroyl-4-methoxy-3,5,5-trimethylcyclohex-3-en-2,6-dione),agglomerone(1-isobutroyl-4-methoxy-5,5-dimethylcyclohex-3-en-2,6-dione),lateriticone(1-valeroyl-4-methoxy-3,5,5-trimethylcyclohex-3-en-2,6-dione),isolateriticone(1-isovaleroyl-4-methoxy-3,5,5-trimethylcyclohex-3-en-2,6-dione andplatyphyllol (6,6-dimethyl-2-acetyl-5-methoxycyclohex-4-ene-1,3-dione),including analogues and derivatives thereof.

Non-limiting examples of tasmanone analogues contemplated by the presentinvention include, but are not restricted to, compounds having thefollowing structural formulae, wherein the structural formula oftasmanone is shown for comparative purposes:

Another preferred subgroup of compounds of formula (I) is represented byformula (V)

wherein

X and Y are independently selected from oxygen, sulfur or —N—R₄; and

A is (C═O)R₁, (C═S)R₁, OR₂, SR₂, (CR₃NR₄R₅), C(R₃)₂OR₂, NR₄R₅,(C═N—R₄)R₁,

N═O, N(═O)₂, NR₄OR₂ or SO₄R₂;

B is as defined above;

C, D, E, F, G and H are each independently selected from H, C₁-C₁₀alkyl, C₂-C₁₀ arylalkyl, C₃-C₆ cycloalkyl, C₂-C₁₀ alkenyl, C₂-C₁₀heteroarylalkyl, C₂-C₁₀ haloalkyl, C₂-C₁₀ dihaloalkyl, C₂-C₁₀trihaloalkyl, C₂-C₁₀ haloalkoxy, OR₂, SR₂, (CR₃NR₄R₅), NR₄R₅,(C═N—R₄)R₁, N═O, N(═O)₂, NR₄OR₂ or SO₄R₂; and

R₁, R₂, R₂, R₄ and R₅ are as defined above.

More specifically unsaturation, epoxides and thioexpoxides may exist atpositions designated by H (or G) connected to F (or E) or F (or E)connected to C (or D). A four-membered ring forming a part of a bicyclicstructure may exist at positions designated by H (or G) connected to C(or D).

Preferred β-diones represented by formula (V) are angustione(1-acetyl-3,5,5-trimethylcyclohex-2,6-dione), dehydroangustione(1-acetyl-3,5,5-trimethylcyclohex-3-en-2,6-dione) and xanthostemone(1-isobutroyl-5,5-dimethylcyclohex-3-en-2,6-dione), including theiranalogues and derivatives.

By way of example, angustione analogues contemplated by the presentinvention include, but are not restricted to, compounds having thefollowing structural formulae, wherein the structural formula ofangustione is shown for comparative purposes:

Non-limiting examples of dehydroangustione analogues include, but arenot restricted to, compounds having the following structural formulae,wherein the structural formula of dehydroangustione is shown forcomparative purposes:

Non-limiting examples of xanthostemone analogues include, but are notrestricted to, compounds having the following structural formulae,wherein the structural formula of xanthostemone is shown for comparativepurposes:

Derivatives of the above compounds include, but are not restricted to,ethoxylate derivatives, propoxylate derivatives, hydrates, aldehydederivatives, ester derivatives, ether derivatives, alcohol derivatives,phenol derivatives, amine derivatives, other biologically or chemicallyequivalent substances, and any combination of two or more of theforegoing.

Similarly effective as pesticides are salts of the above compounds,including mono-valent salts (e.g., sodium, potassium) and di-valentmetal salts (e.g., calcium, magnesium, iron or copper) and ammoniumsalts (e.g., isopropyl ammonium, trialkyl and tetraalkylammonium salts).

The compounds according to any one of formulae (I)-(V) can be preparedaccording to methods analogous to those known in the art for thepreparation of β-diones. Exemplary methods are disclosed for example inEP-A-338992, EP-A-336898, U.S. Pat. No. 4,202,840, U.S. Pat. No.4,869,748, EP-A-186118, EP-A-186119, EP-A-186120, U.S. Pat. No.4,695,673, U.S. Pat. No. 4,780,127, U.S. Pat. No. 4,921,526, U.S. Pat.No. 5,006,150, U.S. Pat. No. 5,545,607, U.S. Pat. No. 5,925,795, U.S.Pat. No. 5,990,046, U.S. Pat. No. 6,218,579, EP-A-249150, EP-A-137963,EP-A-394889 EP-A-506907 or EP-B-135191.

More particularly, compounds according to formulae (III)-(V) can besynthesised using the representative procedures outlined below.

For compounds according to formula (III), 1,3,5-trihydroxybenzene 1 isreacted with CH₃CN in the presence of zinc chloride and hydrochloricacid, according to A. H. Blatt (1943, Org. Synth. Col. II, 522-523),affording 1-acetyl-2,4,6-trihydroxybenzene 2 (phloroacetophenone) (R=Me)(Scheme 1).

R groups other than methyl are depicted above. Reaction of1-acetyl-2,4,6-trihydroxybenzene 2 affording1-acetyl-3,3,5,5-tetramethylcyclohexan-2,4,6-trione 3 is arepresentative procedure for all compounds according to formula (III)(R. A. Gray et al., U.S. Pat. No. 4,202,840) (Scheme 2). Anhydrous MeI(6 eq) is slowly added, at room temperature under an atmosphere ofnitrogen, to a mechanically stirred solution of1-acetyl-2,4,6-trihydroxybenzene 2 (1 eq) and sodium ethoxide (6 eq) inanhydrous methanol. The mixture is refluxed for 4 hours. On cooling themixture is concentrated under vacuo, providing a residue, which isdiluted with water and acidified with 2 M hydrochloric acid.Diethylether extracts are washed with saturated sodium sulfite solution,water and then dried (Na₂SO₄). Evaporation of the diethylether providesthe desired product 3.

For mono, di, tri and tetra B, C, D, E, F substitution patterns,reactions between one and seven mole equivalents of R-I and sodiumethoxide are used. Lawasson's reagent is used for conversion of oxygeninto sulfur groups and sodium borohydride or sodium cyanoborohydride isused to reduce ketone, thioketone and imino groups. When additionalcarbonyl groups are introduced into the cyclohexane ring system theprocedure of Crow is utilised (M. L. Bolte et al., 1985, Agric. Biol.Chem., 49, 761).

Compounds of formula (IV) can be prepared according to a firstrepresentative procedure, as follows:3-methoxy-2,4,4-trimethylcyclohex-2-en-1,5-dione 4 (1 mole eq), preparedaccording to Herzig (J. Herzig, and F. Wenzel, Monatsh, 1903, 24, 101),is dissolved in anhydrous diethylether and hexamethylphosphoramide(solvent ratio, 20:1 respectively) under an atmosphere of nitrogen. Themixture is cooled to 0° C. and lithium hydride (1.1 mole eq) (60% inmineral oil) is added in portions. After addition the mixture is stirredfor a further 10 mins before the addition of benzoyl cyanide 5 [R—CO—CN,R is depicted above] (1.1 mole eq). The mixture is allowed to warm toroom temperature over 12 h at which time the reaction is quenched withwater and partitioned. The ether layer is dried (Na₂SO₄) and evaporatedaffording crude1-benzoyl-3-methoxy-2,4,4-trimethylcyclohex-2-en-1,5-dione 6 which ispurified by SiO₂ column chromatography (hexane/ethyl acetate, gradient)(Scheme 3).

Alternatively, compounds of formula (IV) can be prepared according to asecond representative procedure, as follows:3-methoxy-2,4,4-trimethylcyclohex-2-en-1,5-dione 4 (1 mole eq)(commercially available) and benzoyl cyanide are dissolved in anhydrousdichloromethane and cooled to 0° C. under an atmosphere of nitrogen. Tothe cooled solution is added anhydrous finely powdered zinc chloride(1.1 mole eq.) followed by slow addition of triethylamine (1.2 mole eq).The reaction mixture is stirred at room temperature for 5-6 h and thenpoured into 2 M hydrochloric acid. The mixture is partitioned and thedichloromethane layer is washed with 5% sodium carbonate. The aqueouscarbonate phase is then acidified with hydrochloric acid and extractedwith methylene chloride and dried (Na₂SO₄). The solvent is removed andthe residue subjected to SiO₂ column chromatography (hexane/ethylacetate) affording1-benzoyl-3-methoxy-2,4,4-trimethylcyclohex-2-en-1,5-dione 6 (W. J.Michaely and G. W. Kraatz, EP-B-135191).

Compounds of formula (V) can be prepared according to a firstrepresentative procedure, as follows: 4,4-dimethylcyclohexane-1,3-dione7 (1 mole eq) (commercially available) is dissolved in anhydrousdiethylether and hexamethylphosphoramide (solvent ratio, 20:1respectively) under an atmosphere of nitrogen. The mixture is cooled to0° C. and lithium diisopropylamide (2.1 mole eq) is added dropwise over40 mins. The mixture is then stirred for a further 10 mins before theaddition of methyl iodide (1 mole eq). The mixture is stirred for 12 hand then benzoyl cyanide (2 mole eq) is added and the mixture stirredfor a further 24 h. The reaction was quenched with water and the etherlayer partitioned and dried (Na₂SO₄). The solvent was removed and theresidue subjected to SiO₂ column chromatography (hexane/ethyl acetate)affording 1-benzoyl-3,3,5-trimethylcyclohexan-2,6-dione 8 (Scheme 4).

Alternatively, the compounds of formula (V) can be prepared according toa second representative procedure, as follows:4,4-dimethyl-1,3-cyclohexanedione 7 (1 mole eq) (commercially available)and benzoyl cyanide are dissolved in anhydrous dichloromethane andcooled to 0° C. under an atmosphere of nitrogen. To the cooled solutionis added anhydrous finely powdered zinc chloride (1.1 mole eq) followedby slow addition of triethylamine (1.2 mole eq). The reaction mixture isstirred at room temperature for 5-6 h and then poured into 2 Mhydrochloric acid. The mixture is partitioned and the dichloromethanelayer washed with 5% sodium carbonate. The aqueous carbonate phase isthen acidified with hydrochloric acid and extracted with methylenechloride and dried (Na₂SO₄). The solvent is removed and the residuesubjected to SiO₂ column chromatography (hexane/ethyl acetate) affording1-benzoyl-3,3,5-trimethylcyclohexan-2,6-dione 8. (W. J. Michaely and G.W. Kraatz, EP-B-135191).

Dehydroangustione and xanthostemone derivatives are simply derived fromdehydrogenation of angustione derivatives, for example, by treatment of1-benzoyl-3,3,5-trimethylcyclohexan-2,6-dione 8 with palladium oncharcoal in methanol, which thereby affords1-benzoyl-3,5,5-trimethylcyclohex-3-en-2,6-dione 9 (Scheme 5).

Metal salts (enolates) of the above compounds can be prepared by thereaction of triketone derivatives with the corresponding metalhydroxides suspended in methanol or ethanol. Trialkylammonium salts canbe prepared by the reaction of triketone derivatives (e.g., 3) withtrialkylamines in a chlorinated solvent such as dichloromethane.Tetraalkylammonium salts can be prepared by adding a halogenatedtetraalkylammonium salt to a metal salt in dichloromethane, whichprecipitates the metal halide removed by filtration. The pure materialis obtained by evaporation of the filtrate.

The present inventors have discovered that the β-diones of the inventioncan be obtained from natural sources and, in particular, from volatileoil-bearing organisms. Accordingly, in another aspect, the presentinvention encompasses the use of a β-dione compound, particularly aβ-diketone or a β-triketone compound, obtainable from a volatileoil-bearing organism, including an analogue or derivative thereof, inthe preparation of a pesticidal composition for controlling harmful,annoying or undesired pests.

The present invention contemplates the use of any volatile oil-bearingorganism that produces β-diones, preferably the β-diones according toany one of formulae (I)-(V), and especially (3-diketones and/orβ-triketones, for the preparation of the pesticidal compositions of theinvention. Preferred volatile oil-bearing organisms are volatileoil-bearing plants including, but not restricted to, plants from thefamilies Alliaceae, Apiaceae, Asteraceae, Cannabinaceae, Lamiaceae,Pteridaceae, Myrtaceae, Myoporaceae, Proteaceae, Rutaceae andZingiberaceae. Preferably, the volatile oil-bearing plant is selectedfrom genera of the Myrtaceae family including, but not limited to,Angophora, Austromyrtus, Backhousia, Baeckea, Callistemon, Corymbia,Darwinia, Eucalyptus, Kunzea, Leptospermum, Melaleuca, Syzygium andXanthostemon.

Thus, the compositions of the present invention may contain as activeingredients substantially purified β-diones or crude β-dione-containingextracts obtained from a volatile oil-bearing organism, preferably avolatile oil-bearing plant. Volatile oils, also known in the art asessential oils, typically comprise a volatile mixture of esters,aldehydes, alcohols, ketones and terpenes, which can be prepared frombotanical materials or plant cell biomass from cell culture. Volatileoils can be prepared by subjecting botanical materials to a distillationprocess, for example. A number of different procedures can be used fordistillation. For example, plant matter (e.g., foliage, stems, roots,seeds, bark etc) of a volatile oil-bearing plant is placed in a suitablestill and steam distillation is used to break down the cells of theplant to release the oil. The steam is then condensed and the oil phaseis separated from the aqueous phase to obtain the volatile oil. It willbe appreciated that other methods of volatile oil extraction (e.g.,solvent extraction) are known to those of skill in the art and it willbe understood, in this regard, that the present invention is not limitedto the use or practice of any one particular method of extractingvolatile oils.

Suitably, the compositions comprise naturally-occurring compoundsderived from a volatile oil-bearing organism. Thus, in a preferredembodiment, the pesticidal composition of the invention comprises one ormore β-dione active compounds, particularly β-diketone- and/orβ-triketone-active compounds, that are derived from the volatile oil ofa volatile oil-bearing organism. In this embodiment, the composition mayoptionally contain a naturally-occurring carrier and/or othernaturally-occurring additives.

Naturally-occurring additives contemplated by the present inventioninclude natural antioxidants, which can be used advantageously to reducethe effect of oxidation of the compounds of the invention. An example ofa suitable naturally-occurring antioxidant is α-tocopherol. Otheradditives, such as naturally-occurring stabilisers, are alsocontemplated, which may desirably be added to improve the stability andshelf life of the composition. Examples of suitable natural stabilisersinclude gum arabic, guar gum, sodium caseinate, polyvinyl alcohol,locust bean gum, xanthan gum, kelgum, and mixtures thereof.

In an alternate embodiment, the naturally-occurring compounds derivedfrom a volatile oil may be modified or derivatised to improve, forinstance, their shelf-life, stability, activity and/or bioavailability.

The compounds of the present invention are useful for controllingharmful, annoying or undesired pests. They may be used singularly or incombination with other pest-controlling compounds of the invention. By“controlling” is meant preventing, combating, eradicating, destroying,repelling, or mitigating pests or increasing the mortality or inhibitingthe growth and/or development of pests. The term “pest” is used hereinin its broadest sense and includes within its scope insects, arachnids(e.g., acari, spiders), helminths (e.g., nematodes), molluscs, protozoa(e.g., Plasmodium sp. Paramecium sp.), viruses (e.g., herpesviruses) andthe like. Suitable applications for such control include, but are notlimited to, combating and/or eradicating infestations by pests inanimals (including humans) and/or plants (including trees) and/or storedproducts, which includes the administration to the animal or site of aneffective quantity of a compound of the invention.

By “effective amount” is meant the administration or application of thatamount of active compound, either in a single dose or as part of aseries, that is effective for controlling a significant number of pests.Thus, for example, a “pesticidally-effective” amount is the amount ofactive compound that is effective for increasing the mortality ordecreasing the growth of a significant number of pests. Alternatively, a“pest-repelling” effective amount is the amount of active compound thatis noxious to, and/or induces behavioural changes in, a significantnumber of pests. The effective amount will vary depending upon thetaxonomic group of pest exposed to the active compound, the formulationof the composition, and other relevant factors. It is expected that theamount will fall in a relatively broad range that can be determinedthrough routine trials.

Accordingly, the compounds of the present invention can be used aspesticides, such as but not limited to insecticides, arachnicides,anti-helminthics, molluscicides antivirals, antiprotozoals and the like,or as pest repellents including repellents of insects, arachnids,helminths, molluscs, protozoa and viruses. In especially preferredembodiments, the compounds of the present invention are used in thecontrol of insects, arachnids, helminths or molluscs. In practice, thecompounds can be applied as formulations containing the variousadjuvants and carriers known to or used in the industry for facilitatingbioavailability, stability and dispersion. The choice of formulation andmode of application for any given compound may affect its activity, andselection will be made accordingly.

In general, a pest-controlling compound of the invention can becompounded with appropriate inert carriers and additives in anappropriate ratio by means of dissolving, separating, suspending,mixing, impregnating, adsorbing or precipitating operation to formulateinto oil formulations, emulsifiable concentrates, wettable powders,flowables, granules, powders, dusts, solutions, suspensions, emulsions,controlled-release forms such as microcapsules, aerosols or fumigants.Typically, the compounds of the present invention can be mixed with asolid carrier, liquid carrier or gas carrier, optionally together with asurfactant and other adjuvants useful for such formulations.

The compounds of the invention can be used in an amount from about0.00005% to about 90% by weight as contained in these formulations astheir active component. As used herein, the term “about” refers to aquantity, level, value or amount that varies by as much as 30%,preferably by as much as 20%, and more preferably by as much as 10% to areference quantity, level, value or amount.

Where the compounds are in the form of β-dione-containing extracts, theformulations will usually comprise as their principal active ingredientfrom about 0.0001% to about 90%, preferably from about 0.0001% to about50%, more preferably from about 0.0005% to about 10%, even morepreferably from about 0.0005% to about 5%, even more preferably fromabout 0.0005% to about 1% and still even more preferably from about0.001% to about 0.5% by weight of the extract.

Alternatively, where the compounds are in the form of substantiallypurified preparation of β-diones, the formulations will usually compriseas their principal active ingredient from about 0.00005% to about 90%,preferably from about 0.0001% to about 50%, more preferably from about0.0005% to about 10%, even more preferably from about 0.001% to about 5%and still even more preferably from about 0.001% to about 1% by weightof the substantially purified β-dione.

By “substantially purified” is meant a compound which has been separatedfrom components that naturally accompany it. Typically, a compound issubstantially pure when at least 60%, more preferably at least 75%, morepreferably at least 90%, and most preferably at least 99% of the totalmaterial (by volume, by wet or dry weight, or by mole percent or molefraction) in a sample is the compound of interest. Purity can bemeasured by any appropriate method, e.g., by chromatography or HPLCanalysis.

Examples of solid carriers useful in preparing the formulations areclays including kaolin clay, diatomite, water-containing syntheticsilicon oxide, bentonite, Fubasami clay, and acid clay; talcs; ceramics;inorganic minerals such as Celite, quartz, sulfur, active carbon,calcium carbonate and hydrated silica; and chemical fertilisers such asammonium sulfate, ammonium phosphate, ammonium nitrate, urea andammonium chloride, these solid carriers being finely divided orgranular. Examples of useful liquid carriers are water, alcohols such asmethanol and ethanol, ketones such as acetone and methyl ethyl ketone,aromatic hydrocarbons such as benzene, toluene, xylene, ethylbenzene andmethylnaphthalene, aliphatic hydrocarbons such as hexane, cyclohexane,kerosene and light oil, esters such as ethyl acetate and butyl acetate,nitriles such as acetonitrile and isobutyronitrile, ethers such asdiisopropyl and dioxane, acid amides such as N,N-dimethylformamide andN,N-dimethylacetamide, halogenated hydrocarbons such as dichloromethane,trichloroethane and carbon tetrachloride, dimethyl sulfoxide, and fishoils, mineral oils, plant derived oils such as canola oil, cotton-seedoil, soybean oil and sesame oil as well as essential oils such aslavender oil, eucalyptus oil, tea tree oil, citrus oil etc. Solid orliquid carriers can be used alone or in combination. Examples of gascarriers, i.e., those of propellants, are butane gas, LPG (liquefiedpetroleum gas), dimethyl ether, fluorocarbons and carbon dioxide gas.

Examples of surfactants are alkylsulfuric acid esters, alkylsulfonicacid salts, alkylarylsulfonic acid salts, alkyl aryl ethers andpolyoxyethylene adducts thereof, polyethylene glycol ethers, polyhydricalcohol esters, sugar alcohol derivatives, sorbitane monolaurate,alkylallyl sorbitane monolaurate, alkylbenzene sulfonate,alkylnaphthalene sulfonate, lignin sulfonate, and sulfuric acid estersalts of higher alcohols. These surfactants may be used alone or incombination.

Examples of adjuvants for the formulations, such as binders anddispersants, are casein, gelatin, polysaccharides such as starch, gumarabic, cellulose derivatives and alginic acid, lignin derivatives,bentonite, sugars and water-soluble synthetic high-molecular-weightsubstances such as polyvinyl alcohol, polyvinyl pyrrolidone andpolyacrylic acids. Examples of stabilisers are PAP (acid isopropylphosphate), BHT (2,6-di-tert-butyl-4-methylphenol), BHA (mixture of2-tert-butyl-4-methoxyphenol and 3-tert-butyl-4-methoxyphenol),synergists such as piperonyl butoxide, vegetable oils, mineral oils,fish oils, surfactants and fatty acids or esters thereof.

Emulsifying agents that may be used are suitably one or more of thoseselected from non-ionic or anionic emulsifying agents. Examples ofnon-ionic emulsifying agents include, but are not restricted to,polyoxyethylenealkylphenylether, polyoxyethylenealkylether,polyethyleneglycol fatty ester, sorbitan fatty ester, polyoxyethylenesorbitan fatty ester, polyoxyethylenesorbitol fatty ester,polyoxyethylenepolyoxypropylenealkylether. Examples of anionicemulsifying agents include alkyl sulphates, polyoxyethylenealkylethersulphates, sulfosuccinates, taurine derivatives, sarcosine derivatives,phosphoric esters, alkylbenzenesulfonates and the like. A mixtureconsisting of polyoxyethylenestyrylphenylether and calciumallylbenzenesulfonate is preferred. These emulsifying agents may be usedin an amount of 5 to 20 weight parts per 100 weight parts of thecompositions of the present invention.

Formulation thus obtained can be used solus or diluted, for example,with water or other diluent. The formulations can be used also asadmixtures with other pesticides such as insecticides, arachnids,anti-helminthics, molluscicides, herbicides, plant growth regulators,synergists, soil improvers, baits and the like, or can be usedsimultaneously with such agents without mixing. For example, thepest-controlling compounds of the invention can be combined with othernaturally derived bioactive compounds or extracts such as neem or itscomponents, derris, pyrethrum; microbial extracts such as avermectins orstreptomycins; with synthetic insecticides, acaricides, molluscicides,anti-helminthics; antiprotozoals, antivirals or with microorganismshaving insecticidal, acaricidal, molluscicidal, anti-helminthicanti-protozoal or antiviral activity e.g., bacteria such as Bacillusthuringiensis, Bacillus popillae, entomogenous fungi such as Metarhiziumspp., Verticillium lecanii, nematodes such as Steinernema spp andHeterorhabditis. Alternatively, or in addition, the pest-controllingcompounds of the invention can be combined with synergists such aspiperonyl butoxide, and with ultraviolet screening compounds of naturalor synthetic origin.

When used as an agricultural pesticide, the compound of the invention ispreferably applied usually in an amount of 0.01 to 500 g/100 m². When anemulsifiable concentrate, wettable powder or flowables are used asdiluted with water, the compound is applied usually at a concentrationof 0.1 to 1000 ppm, preferably 1 to 500 ppm. The granular or dust can beapplied without dilution.

The amount or concentration of application, although exemplified above,can be suitably increased or reduced according to the type ofpreparation, time, place, method of application, kind of pest and extentof harm or annoyance suffered.

The invention also contemplates the use of the above described β-dionecompounds in pest repellent, particularly insect repellent,compositions. Repellent compositions contemplated by the presentinvention include those that are noxious to, and/or induce behaviouralchanges in, a pest. The latter compositions suitably comprise anactivity including, but not restricted to, an antifeedant activity, anoviposition deterrent activity and an insect growth regulatory activity.Insect repellent compositions in various dosage forms can be prepared byblending the above-described β-dione compounds as active ingredientswith a base of cosmetics or pharmaceuticals, which are usually appliedto human bodies or animals. They can be formulated in, for example,lotions, aerosols, milky lotions, creams or the like. These compoundscan be further incorporated with other insect repellents, antioxidants,UV-absorbers, humectants or other additives.

The above compounds or the above-prepared compositions of the presentinvention can be applied directly to human bodies or animals. Besides,substrates, such as sheets, films, nets, timber or the like, which havepreliminarily been treated with the above compounds or compositions bymeans of application, impregnation or blending, can also be used.

The quantity of the above compounds to be formulated in thenoxious-insect repellents depends upon the dosage form, usage or otherconditions. Suitable dosages may be selected from about 0.1% to about90% by weight.

Thus, in another aspect of the present invention there is provided amethod for controlling harmful, annoying or undesired pests, said methodcomprising exposing said pests to a pest-controlling effective amount ofa composition comprising a β-dione compound as broadly described above.Preferred embodiments of this type include exposing said pests to apesticidally effective amount or a pest-repelling effective amount ofsaid composition.

The terms “comprise”, “comprises” and “comprising” and the like refer,unless the context requires otherwise, to the inclusion of a stated stepor element or group of steps or elements but not the exclusion of anyother step or element or group of steps or elements.

The compositions and methods of the present invention may be applied topests including, but not restricted to, insects, arachnids, helminths,molluscs, protozoa and viruses. For example, suitable insects that fallwithin the scope of the present invention include those:

(a) from the order of the lepidopterans (Lepidoptera), for example,Adoxophyes orana, Agrotis ipsilon, Agrotis segetum, Alabama argillacea,Anticarsia gemmatalis, Argyresthia conjugella, Autographa gamma,Cacoecia murinana, Capua reticulana, Choristoneura fumiferana, Chilopartellus, Choristoneura occidentalis, Cirphis unipuncta, Cnaphalocrocismedinalis, Crocidolomia binotalis, Cydia pomonella, Dendrolimus pini,Diaphania nitidalis, Diatraea grandiosella, Earias insulana,Elasmopalpus lignosellus, Eupoecilia ambiguella, Feltia subterranea,Grapholitha funebrana, Grapholitha molesta, Heliothis armigera,Heliothis virescens, Heliothis zea, Hellula undalis, Hiberniadefoliaria, Hyphantria cunea, Hyponomeuta malinellus, Keiferialycopersicella, Lambdina fiscellaria, Laphygma exigua, Leucopterascitella, Lithocolletis blancardella, Lobesia botrana, Loxostegesticticalis, Lymantria dispar, Lymantria monacha, Lyonetia clerkella,Manduca sexta, Malacosoma neustria, Mamestra brassicae, Mocis repanda,Operophthera brumata, Orgyia pseudotsugata, Ostrinia nubilalis, Pandemisheparana, Panolis flammea, Pectinophora gossypiella, Phthorimaeaoperculella, Phyllocnistis citrella, Pieris brassicae, Plathypenascabra, Platynota stultana, Plutella xylostella, Prays citri, Praysoleae, Prodenia sunia, Prodenia ornithogalli, Pseudoplusia includens,Rhyacionia frustrana, Scrobipalpula absoluta, Sesamia inferens,Sparganothis pilleriana, Spodoptera frugiperda, Spodoptera littoralis,Spodoptera litura, Syllepta derogata, Synanthedon myopaeformis,Thaumatopoea pityocampa, Tortrix viridana, Trichoplusia ni, Tryporyzaincertulas, Zeiraphera canadensis,

(b) furthermore Galleria mellonella and Sitotroga cerealella, Ephestiacautella, Tineola bisselliella;

(c) from the order of the beetles (Coleoptera), for example, Anthonomusgrandis, Anthonomus pomorum, Apion vorax, Atomaria linearis,Blastophagus piniperda, Cassida nebulosa, Cerotoma trifurcata,Ceuthorhynchus assimilis, Ceuthorhynchus napi, Chaetocnema tibialis,Conoderus vespertinus, Crioceris asparagi, Dendroctonus refipennis,Diabrotica longicornis, Diabrotica 12-punctata, Diabrotica virgifera,Epilachna varivestis, Epitrix hirtipennis, Eutinobothrus brasiliensis,Hylobius abietis, Hypera brunneipennis, Hypera postica, Ips typographus,Lema bilineata, Lema melanopus, Leptinotarsa decemlineata, Limoniuscalifornicus, Lissorhoptrus oryzophilus, Melanotus communis, Meligethesaeneus, Melolontha hippocastani, Melolontha melolontha, Oulema oryzae,Ortiorrhynchus sulcatus, Otiorrhynchus ovatus, Phaedon cochleariae,Phyllopertha horticola, Phyllophaga sp., Phyllotreta chrysocephala,Phyllotreta nemorum, Phyllotreta striolata, Popillia japonica,Psylliodes napi, Scolytus intricatus, Sitona lineatus,

(d) furthermore Bruchus rufimanus, Bruchus pisorum, Bruchus lentis,Sitophilus granaria, Lasioderma serricorne, Oryzaephilus surinamensis,Rhyzopertha dominica, Sitophilus oryzae, Tribolium castaneum, Trogodermagranarium, Zabrotes subfasciatus;

(e) from the order of the dipterans (Diptera), for example, Anastrephaludens, Ceratitis capitata, Contarinia sorghicola, Dacus cucurbitae,Dacus oleae, Dasineura brassicae, Delia coarctata, Delia radicum,Hydrellia griseola, Hylemyia platura, Liriomyza sativae, Liriomyzatrifolii, Mayetiola destructor, Orseolia oryzae, Oscinella frit, Pegomyahyoscyami, Phorbia antiqua, Phorbia brassicae, Phorbia coarctata,Rhagoletis cerasi, Rhagoletis pomonella,

(f) furthermore Aedes aegypti, Aedes vexans, Anopheles maculipennis,Chrysomya bezziana, Chrysomya hominivorax, Chrysomya macellaria,Cordylobia anthropophaga, Culex pipiens, Fannia canicularis,Gasterophilus intestinalis, Glossina morsitans, Haematobia irritans,Haplodiplosis equestris, Hypoderma lineata, Lucilia caprina, Luciliacuprina, Lucilia sericata, Musca domestica, Muscina stabulans, Oestrusovis, Tabanus bovinus, Simulium damnosum;

(g) from the order of the thrips (Thysanoptera), for example,Frankliniella fusca, Frankliniella occidentalis, Frankliniella tritici,Haplothrips tritici, Heliothrips haemorrhoidalis, Scirtothrips citri,Thrips oryzae, Thrips palmi, Thrips tabaci;

(h) from the order of the hymenopterans (Hymenoptera), for example,Athalia rosae, Atta cephalotes, Atta sexdens, Atta texana, Hoplocampaminuta, Hoplocampa testudinea, Iridomyrmes humilis, Iridomyrmexpurpureus, Monomorium pharaonis, Solenopsis geminata, Solenopsisinvicta, Solenopsis richteri, Technomyrmex albipes;

(i) from the order of the heteropterans (Heteroptera), for example,Acrosternum hilare, Blissus leucopterus, Cyrtopeltis notatus, Dysdercuscingulatus, Dysdercus intermedius, Eurygaster integriceps, Euschistusimpictiventris, Leptoglossus phyllopus, Lygus hesperus, Lyguslineolaris, Lygus pratensis, Nezara viridula, Piesma quadrata, Solubeainsularis, Thyanta perditor;

(j) from the order of the homopterans (Homoptera), for example,Acyrthosiphon onobrychis, Acyrthosiphon pisum, Adelges laricis,Aonidiella aurantii, Aphidula nasturtii, Aphis fabae, Aphis gossypii,Aphis pomi, Aulacorthum solani, Bemisia tabaci, Brachycaudus cardui,Brevicoryne brassicae, Dalbulus maidis, Dreyfusia nordmannianae,Dreyfusia piceae, Dysaphis radicola, Empoasca fabae, Eriosoma lanigerum,Laodelphax striatella, Macrosiphum avenae, Macrosiphum euphorbiae,Macrosiphon rosae, Megoura viciae, Metopolophium dirhodum, Myzuspersicae, Myzus cerasi, Nephotettix cincticeps, Nilaparvata lugens,Perkinsiella saccharicida, Phorodon humuli, Psylla mali, Psylla piri,Psylla pyricola, Rhopalosiphum maidis, Schizaphis graminum, Sitobionavenae, Sogatella furcifera, Toxoptera citricida, Trialeurodesabutilonea, Trialeurodes vaporariorum, Viteus vitifolii;

(k) from the order of the termites (Isoptera), for example, Calotermesflavicollis, Coptotermes spp, Leucotermes flavipes, Macrotermessubhyalinus, Nasutitermes spp such as Nasutitermes walkeri, Odontotermesformosanus, Reticulitermes lucifugus, Termes natalensis;

(l) from the order of the orthopterans (Orthoptera), for example,Gryllotalpa gryllotalpa, Locusta migratoria, Melanoplus bivittatus,Melanoplus femur-rubrum, Melanoplus mexicanus, Melanoplus sanguinipes,Melanoplus spretus, Nomadacris septemfasciata, Schistocerca americana,Schistocerca peregrina, Stauronotus maroccanus, Schistocerca gregaria,

(m) furthermore Acheta domestica, Blatta orientalis, Blattellagermanica, Periplaneta americana;

(n) from the order of the phthirapterans (Phthiraptera), for example,Mallophaga, such as Damalina spp., and Anoplura such as Linognathus andHaematopinus spp.;

(o) from the order of the hemipterans (Hemiptera), for example, Aphis,Bemisia, Phorodon, Aeneolamia, Empoasca, Parkinsiella, Pyrilla,Aonidiella, Coccus, Pseudococcus, Helopeltis, Lygus, Dysdercus,Oxycarenus, Nezara, Aleyrodes, Triatoma, Psylla, Myzus, Megoura,Phylloxera, Adelges, Nilaparvata, Nephotettix or Cimwx spp.;

(p) from the order of the siphonapterans (Siphonaptera), for example,Ctenocephalides or Pulex spp.;

(q) from the order of the thysanurans (Thysanura), for example, Lepismaspp.;

(r) from the order of the dermapterans (Dermaptera), for example,Forficula spp.; and

(s) from the order of the psocopterans (Psocoptera), for example,Peripsocus spp.

Arachnids contemplated by the present invention include, but are notlimited to, spiders and scorpions and especially mites such asphytophagous mites (Acari), such as Aculops lycopersicae, Aculopspelekassi, Aculus schlechtendali, Brevipalpus phoenicis, Bryobiapraetiosa, Eotetranychus carpini, Eutetranychus banksii, Eriophyessheldoni, Oligonychus pratensis, Panonychus ulmi, Panonychus citri,Phyllocoptruta oleivora, Polyphagotarsonemus latus, Tarsonemus pallidus,Tetranychus cinnabarinus, Tetranychus kanzawai, Tetranchus pacificus,Tetranychus urticae, ticks, such as Amblyomma americanum, Amblyommavariegatum, Argas persicus, Boophilus annulatus, Boophilus decoloratus,Boophilus microplus, Dermacentor silvarum, Hyalomma truncatum, Ixodesricinus, Ixodes rubicundus, Ornithodorus moubata, Otobius megnini,Rhipicephalus appendiculatus and Rhipicephalus evertsi, andanimal-parasitic mites, such as Dermanyssus gallinae, Psoroptes ovis andSarcoptes scabiei.

Helminths falling within the scope of the present invention may beselected from cestodes such as fish tapeworm, pork tapeworm, beeftapeworm, and dwarf tapeworm; trematodes such as from the generaMetagonimus and Heterophyes; and nematodes such as but not limited tofilariid, ascarid, strongyle and trichostrongyle nematodes of the generaAcanthocheilonema, Aelurostrongylus, Ancylostoma, Angiostrongylus,Ascaris, Brugia, Bunostomum, Dictyocaulus, Dioctophyme, Dipetalonema,Dirofilaria, Dracunculus, Filaroides, Lagochilascaris, Loa, Mansonella,Muellerius, Necator, Onchocerca, Parafilaria, Parascaris,Protostrongylus, Setaria, Stephanofilaria, Strongyloides, Strongylus,Thelazia, Toxascaris, Toxocara, Trichinella, Uncinaria and Wuchereria.

Suitable molluscs include those of the Gastropoda class examples ofwhich include snails, slugs, conchs, and whelks.

Protozoa may be selected for example from Plasmodia, Toxoplasma,Leishmania, Trypanosoma, Giardia, Entamoeba, Acanthamoeba, Nagleria,Hartmanella, Balantidium, Babesia, Cryptosporidium, Isospora,Microsporidium, Trichomonas or Pneumocystis species.

Viruses may be selected from RNA viruses or DNA viruses, which includebut are not limited to Human Immunodeficiency Virus (HIV), Poliovirus,Influenza virus, Rous Sarcoma virus, Flaviviruses such as Japaneseencephalitis, Influenza virus, Respiratory Syncytial Virus, Hepatitisvirus, Parvovirus, Rotavirus, Coronavirus, Adenovirus and Herpesvirusessuch as Papillomavirus and Epstein-Barr virus.

The present invention also extends to methods for producing resistancein plants to pests including, but not limited to, insects, arachnids,helminths, molluscs, protozoa and viruses by crossing a plant expressinga β-dione compound according to the invention with pest susceptiblelines. Crossing a β-dione-producing plant into a pest susceptiblebackground would produce a resistant plant with a high level of pestresistance. Plants that could be made pest resistant include, but arenot limited to, dicotyledonous plants, especially trees and moreespecially members of the Myrtaceae family. For example E. cloezianacommonly known as Gympie Messmate is one of the many Eucalyptus speciesgrown for hard wood production. However the oil present in thischemotype does not contain β-diones and hence an intra species crosswith the unique North Queensland tasmanone chemotype would introducethis phenotypic trait. Such a process would be readily applicable toother Eucalyptus species of commercial interest. Interspecific crossingwithin the Myrtaceae family is well established to those skilled in theart and inclusion of β-dione as an additional trait into formal breedingprograms is acknowledged.

Suitable β-dione-producing plants may be selected from the familiesAlliaceae, Apiaceae, Asteraceae, Cannabinaceae, Lamiaceae, Pteridaceae,Myrtaceae, Myoporaceae, Proteaceae, Rutaceae and Zingiberaceae.Preferably, the volatile oil-bearing plant is selected from genera ofthe Myrtaceae family including, but not limited to, Angophora,Austromyrtus, Backhousia, Baeckea, Callistemon, Corymbia, Darwinia,Eucalyptus, Kunzea, Leptospermum, Melaleuca, Syzygium and Xanthostemon.Preferred 3-dione-producing plants are Leptospermum morrisonii,Eucalyptus bensonii, Eucalyptus megacornuta, Eucalyptus pilularis,Eucalyptus cornuta, Eucalyptus baxteri, Eucalyptus macrorhyncha,Eucalyptus cloeziana, Melaleuca cajuputi, Eucalyptus jensenii,Backhousia angustifolia and Leptospermum scoparium. A particularlypreferred β-dione-producing plant is Eucalyptus cloeziana.

As used herein, the term “plant” includes reference to whole plants,plant organs (e.g., leaves, stems, roots, etc.), seeds and plant cellsand progeny of same. Plant cell, as used herein includes, withoutlimitation, seeds suspension cultures, embryos, meristematic regions,callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen,and microspores. The class of plants which can be used in the methods ofthe invention is generally as broad as the class of higher plantsamenable to transformation techniques, including both monocotyledonousand dicotyledonous plants.

Thus, the present invention contemplates conventional plant breedingmethods to transfer the genetic material associated with β-dioneproduction via crossing and backcrossing. Such methods will comprise thesteps of: (1) sexually crossing the β-dione-producing plant with a plantfrom a pest susceptible taxon; (2) recovering reproductive material fromthe progeny of the cross; and (3) growingβ-dione-producing/pest-resistant plants from the reproductive material.Where desirable or necessary, the agronomic characteristics of thesusceptible taxon can be substantially preserved by expanding thismethod to include the further steps of repetitively: (1) backcrossingthe pest-resistant progeny with pest-susceptible plants from thesusceptible taxon; and (2) selecting for expression of a β-dione (or anassociated marker gene) among the progeny of the backcross, until thedesired percentage of the characteristics of the susceptible taxon arepresent in the progeny along with the gene or genes imparting β-dioneactivity.

By the term “taxon” herein is meant a unit of botanical classification.It thus includes, genus, species, cultivars, varieties, variants andother minor taxonomic groups which lack a consistent nomenclature.

In order that the invention may be readily understood and put intopractical effect, particular preferred embodiments will now be describedby way of the following non-limiting examples.

EXAMPLES Example 1 β-Triketone-Containing Oils Obtained from AustralianMyrtaceae Species

Australia has an extensive number of volatile oils from species of theMyrtaceae that are rich in a diversity of structurally relatedconstituents known as 3-triketones. These oils often show not only ahigh yield of oil, but also a high degree of biosynthetic selectivitythat produces β-triketones in a high proportion of the oil composition.The major constituents of the published Myrtaceae essential oils(Hellyer, 1968; Boland and Brophy, 1990, 1993; Brophy, et al., 1995;Bignall et al., 1997; Southwell and Brophy 2000) are listed in Table 1and their structures are included in FIG. 1.

TABLE 1 β-Triketone Profiles of Australian Essential Oils Species Yieldβ-Triketone (%) Distribn Backhousia angustifolia 2.5 Angustione (85) QLDBackhousia angustifolia 2.5 Dehyroangustione (80) QLD Eucalyptuscloeziana 3.0 Tasmanone (95) QLD Eucalyptus suberea 1.4 Tasmanone (94)WA Eucalyptus lateritica 0.9 Tasmanone (37), lateriticone (14) WAEucalyptus camfieldii Tasmanone (40) NSW Leptospermum scoparium 0.4Leptospermone (19), Flavesone (8), NSW, NZ Isoleptospermone (5)Eucalyptus grandis 0.6 Leptospermone (20), Flavesone (13), NSWIsoleptospermone (3) Eucalyptus agglomerata Agglomerone (40) NSWEucalyptus mckieana Agglomerone (60) NSW Eucalyptus bensonii 2.5Agglomerone (72) Eucalyptus insularis 1 Agglomerone (19) WA Eucalyptusjensenii 0.3 Jensenone (70) NT Eucalyptus papuana 0.7 Papuanone (40) NthAus Leptospermum morrisonii 1.8 Grandiflorone (58) NSW Melaleucacajeputi Platyphyllol Nth Aus Xanthostemon Xanthostemone oppositifolius

The β-triketones obtained from selected Myrtaceous volatile oils wereshown to have significant insecticidal and/or acaricidal activity.

Example 2 Insecticidal Activity

Initial insecticidal screening against two important arthropod species,two-spotted mite (Tetranychus urticae) and diamond back moth (Plutellaxylostella) lavae highlighted three oils on the basis of efficacy, oilyield, oil profile and ease of recollection (Table 2). Where feasible,the LD₅₀ and LD₉₅ values were determined.

TABLE 2 Percentage Mortality of Three Efficacious Oils Two Spotted MiteDiamond Back Moth % Mortality % Mortality Species (0.5%/1.0%)(0.5%/1.0%) Backhousia angustifolia 98/98 100/100 Backhousiaangustifolia 100/100 100/100 Eucalyptus cloeziana 100/100  70/100

Insecticidal tests using the oil from fresh plant recollections andsteam distillations varied occasionally and, in this regard, it isbelieved that improving storage conditions including temperature, lightand exposure to air and inclusion of a dessicant can enhance thestability of the active fraction of such oils.

E. cloeziana oil continued to show high potency against both insecttests. This oil exhibited an LD₉₅ between 0.04-0.20% (depending onformulation and treatment) against two-spotted mite. An LD₉₅ of 0.10%was observed against 1^(st) instar lavae of diamond back moth and thisrose to 0.78% when tested against 3^(rd) instar lavae. In additionalpreliminary investigations with greenhouse thrips (Heliothripshaemorrhoidalis), a 0.1% concentration of E. cloeziana oil induced 100%mortality.

Additional work was carried out on the E. cloeziana oil to explore thecontribution the various components make to the overall efficacy of thisoil. Fractions 1, 3, 4, and 5 outlined in Example 3 were screenedagainst two-spotted mite. Fractions 1 and 5 showed no significantinsecticidal effect. Fractions 3 and 4, comprising 98% and 99% tasmanonewere active and showed little difference to the activity of the wholeoil.

This suggests that not only is tasmanone the principle component in theoil, but it is also the principle bioactive constituent. It is alsoreasonable to assume that as the activity of E. cloeziana oil has beendemonstrated against a number of different arthropod species, namely amite (T. urticae), a caterpillar (P. xylostella) and a thrips (H.haemorrhoidalis), its insecticidal activity is broad in nature.

Example 3 Chemistry

Chemical analysis (GC-MS) of the steam-distilled oils from the collectedplants in this work are summarised in Table 3.

TABLE 3 β-Triketone Profiles of Selected Oils Plant Source PrincipleComponent* (%) Backhousia angustifolia -I Dehydroangustione 85%Backhousia angustifolia -II Dehydroangustinone (80%) Backhousiaangustifolia -III Angustinone (65%) Backhousia angustifolia -IVAngustinone (28%) Eucalyptus cloeziana Tasmanone (84-96%) Melaleucacajuputi subsp platyphylla Platyphyllol (64-71%) * As determined by gaschromatography

The most promising oils were derived from B. angustifolia, M. cajuputisubsp platyphylla and E. cloeziana and these were then subjected toadditional chemical fractionation. The lower insecticidal activityobserved in the recollected B. angustifolia (IV) was in part attributedto the lower levels of β-triketone. The level of β-triketone waselevated by the removal of the more volatile monoterpenes using vacuumdistillation.

One of the most efficacious oils was from E. cloeziana, a tasmanone(84-96%) rich oil with additional terpenes and β-triketones (FIG. 2,Table 4), which displayed consistent activity at every stage ofprocessing and formulation. This oil was, therefore, fractionated usingcolumn chromatography on silica gel with a hexane-diethyl ether gradientand a final methanol elution. The profiles of the fractions aresummarised in Table 5.

TABLE 4 Chemical Profile of E. cloeziana Oil Peak No CompoundComposition (%) 1 a-pinene 0.5-1.9 2 B-pinene 1.5-5.7 3 Limonene 0.1-0.64 a-terpineol 0.7-2.0 5 Globulol 0.01-0.5  6 Agglomerone 0.01-0.6  7Tasmanone 84-96 8 Lateriticone 0.2-0.7 9 Isolateriticone 0.3-1.2

TABLE 5 Fractions Cut From E. cloeziana Oil Fraction No Solvent SystemComposition Amount F1 Hexane Hydrocarbons 73 mg F2 Hex:Et2O (9:1) 80%Tasmanone 4 mg F3 Hex:Et2O (1:1) 98% Tasmanone 3.66 g F4 Hex:Et2O (9:1)+99% Tasmanone 694 mg F5 MeOH Terpene alcohols 64 mg

¹H NMR data (FIG. 3) were recorded on F4 and confirmed the structure oftasmanone. Moreover the compound exists in solution (CDCl₃) intautomeric forms (FIG. 4) in the ratio 2:0:1 (A:B:C).

Another efficacious oils was from M. cajuputi subsp platyphylla, aplatyphyllol (64-71%) rich oil (Table 6), which also displayedconsistent activity at every stage of processing and formulation.

TABLE 6 Typical Chemical Profile of M. cajuputi subsp platyphylla OilPeak No Compound Composition (%) 1 a-pinene  Tr-0.8 2 1,8-cineole Tr-0.7 3 B-caryophyllene 0.6-3.2 4 Humulene 0.6-1.3 5 Spathulenol4.0-9.0 6 Caryophyllene oxide  Tr-3.6 6 Platyphyllol 64-71 7 MW 234 -unknown 4.3 *As determined by gas chromatography

Example 4 Phytotoxicity

Initial investigations using leaves and leaf discs of several plantspecies including French bean (Phaseolus vulgaris) lemon (Citrus limon)Orange (Citrus sinensis) and Cabbage (Brassica oleracea) indicated thatphytotoxicity did not occur for most oils below 0.5%. More detailedinvestigations using soft intact leaves of young greenhouse-grown Frenchbean subsequently showed E. cloeziana oil applied as a spray caused somephytotoxicity at concentrations of 0.5% and above.

Example 5 Toxicity of E. cloeziana Oil Bacterial Reverse Mutation Assay

This study investigated the potential of E. cloeziana oil to inducereverse mutations at the histidine locus in the genome of one strain ofSalmonella typhimurium TA100 in the presence and absence of a metabolicactivation system (mammalian microsomal enzymes, S9 mix). The testsample was dissolved in dimethyl sulfoxide (DMSO). In this assay, an E.cloeziana oil test sample did not induce an appropriate-fold increase (a2-fold increase for TA100) in the mean revertants per plate in thetester strain TA100 over the mean revertants per plate of theappropriate vehicle control. Accordingly, the test sample was consideredto be non-mutagenic under the conditions of the assay.

Acute Oral Sighting

The acute oral toxicity of E. cloeziana oil was investigated in ten (10)Sprague Dawley Specific Pathogen Free female rats (groups of 2) at dosesof 500, 250, 125 and 50 mg/kg. The experimental procedure was based onOECD guidelines for the testing of chemicals, No. 420.

Clinical signs of toxicity occurred between one (1) and twenty-four (24)hours after dosing. Both animals in the 500 mg/kg group exhibitedsubdued behaviour, partial eye closure, slow breathing, reduced motoractivity, ataxia followed by death within 24 hours. The animals in the250 mg/kg group exhibited subdued behaviour, partial eye closure, slowbreathing, social isolation, and reduced motor activity, and hadreturned to normal by 24 hours after dosing. The animals in the 125mg/kg group exhibited subdued behaviour, partial eye closure, slowbreathing, piloerection and reduced motor activity, and had returned tonormal by 24 hours after dosing. The animals in the 50 mg/kg group didnot show any signs of toxicity during the seven day experimental period.

There were no other clinical abnormalities in any animal throughout theseven (7) day observation period. The stomach of one animal in the 250mg/kg group (5F) had a single ulcer. There were no other grossabnormalities in the major organs of any animal at autopsy.

Based on the results obtained from this study 50, 100 and 200 mg/kg canbe selected for a main study as the maximum non-toxic dose, intermediatedose and high dose, respectively.

Full Study

The acute oral toxicity of E. cloeziana oil was investigated in thirty(30) Sprague Dawley Specific Pathogen Fee rats (15 males and 15 females)at doses of 50, 100 and 200 mg/kg. These doses were chosen following adose range finding in the above study. The experimental procedure wasbased on OECD guidelines for the testing of chemicals, No. 420.

The acute NOAEL of E. cloeziana oil was determined to be 50 mg/kg, andthe MTD was 200 mg/kg under the conditions of this study.

Acute Dermal

The acute dermal toxicity of E. cloeziana oil was investigated in ten(10) Sprague Dawley Specific Pathogen Free rats (5 males and 5 females)at a dose of 2000 mg/kg. A preliminary study (SIGHTING) indicated nosigns of toxicity at this dose. The experimental procedure was based onOECD guidelines for the testing of chemicals, No. 420.

No clinical abnormalities, skin irritations or body weight losses wereobserved in any animal throughout the fourteen (14) day observationperiod. No deaths occurred. No abnormalities were seen in the majororgans at necropsy. The rat acute dermal LD50 of E. cloeziana oil wasdetermined to be greater than 2000 mg/kg under the conditions of thisstudy.

Skin Irritancy/Corrosion

The potential of a test sample of E. cloeziana oil to provoke skinirritation/corrosion reactions was investigated using a primary dermalirritation/corrosion test in three (3) New Zealand White albino rabbits(OECD Guidelines for the Testing of Chemicals, No. 404). The resultsobtained from this study indicated that E. cloeziana oil is anon-irritant according to the National Occupational Health and SafetyCommission (NOHSC) “Approved Criteria for Classifying HazardousSubstances [NOHSC:1008 (1999)]”.

Example 6 Isolation and Purification of β-Triketones from AustralianMyrtaceae Species

Plant collections were commissioned at the Mt. Annan Botanic Garden,Sydney, Australia, and the Darwin Botanic Garden, Northern Territory,Australia to provide potential sources of a range of β-triketoneisolation. The plants investigated for β-triketone exploration arelisted in Table 6.

TABLE 6 Plants Sourced for β-Triketone Exploration Accession β-triketone# Source Plant Plant Location # anticipated 1 Leptospermum morrisoniiMt. Annan, NSW 873247 grandiflorone 2 Eucalyptus bensonii Mt. Annan, NSW881045 agglomerone 3 Eucalyptus megacornuta Mt. Annan, NSW 831078jensenone ⁺ 4 Eucalyptus pilularis Mt. Annan, NSW 873166 torquatone 5Eucalyptus cornuta Mt. Annan, NSW 852618 jensenone ⁺ 6 Eucalyptusbaxteri Mt. Annan, NSW 860999 agglomerone/ tasmanone 7 Eucalyptusmacrorhyncha Mt. Annan, NSW 860896 conglomerone 8 Eucalyptus cloezianaLappa, QLD PF 2513 tasmanone 9 Melaleuca cajuputi ssp Bensbach, WP, PNGKW16-19 platyphyllol platyphyla 10 Eucalyptus jensenii NT BotanicGardens, RK114- jensenone Darwin, NT LUS 11 Backhousia angustifoliaWilgavale, Texas, PF1712 dehydroangustione QLD 12 Backhousiaangustifolia Didcott Creek, QLD PF 1708 angustione 13 Leptospermumscoparium NA commercial oil NA flavesone, sample isoleptospermone,leptospermone 14 Eucalyptus conjuncta Mt Annan 854097 conglomerone ⁺ Oilcontains other β-triketone constituents

All plants, apart from 9 and 13, were steam distilled to yield variousquantities of essential oil. All oils were analysed by GasChromatography Mass Spectrometry (GCMS) to determine the presence andabundance of β-triketones. Based on this information, particular oilswere targeted for isolation and purification of β-triketones using wetchemistry and preparative HPLC techniques.

The β-triketones listed in Table 7 were isolated in quantities adequatefor insecticidal screening. A minimum of 50 mg of each compound wasrequired. Isoleptospermone and leptospermone were difficult to separatedue to their structural similarity as were angustione anddehydroangustione. Consequently, mixtures of these compounds, where oneisomer was significantly more abundant, were provided for insecticidalscreening as this will still allow for differentiation in observedactivity.

TABLE 7 β-Triketones Isolated for Insecticidal Screening Amount sentSample Purity % by for screening β-triketones Plant source GCMS (mg)grandiflorone Leptospermum morrisonii 100.00  99.5 jensenone Eucalyptusjensenii 100.00  67.9 dehydroangustione Backhousia angustifolia 95.02165.02 PF1712 angustione Backhousia angustifolia 66.30 (33.70% 107.1PF1708 dehydroangustione) agglomerone Eucalyptus bensonii 99.28 109.2flavesone Leptospermum scoparium 99.32 101.8 isoleptospermoneLeptospermum scoparium 33.10 (66.90% 113.4 leptospermone) leptospermoneLeptospermum scoparium 95.33 77.0 tasmanone Eucalyptus cloeziana 99.92110.1 platyphyllol Melaleuca cajuputi subsp. 99.62 255.6 platyphylla

The chemical structures and identities of the β-triketones isolated wereconfirmed by GCMS and Nuclear Magnetic Resonance (NMR) analysis.

Example 7 Efficacy of E. cloeziana Oil on Target Organisms TargetOrganisms

Two spotted mite (TSM) Tetranychus urticae Koch [Acarina: Tetranychidae]were collected from a mass culture maintained at the University ofWestern Sydney's Hawkesbury Campus in Richmond, NSW, Australia. Theywere reared on potted French beans (Phaseolus vulgaris L [Fabales:Fabaceae] in a glasshouse maintained at 25+5° C., 65±5% RH and 14 h D:Lphotoperiod. Only young females were selected for bioassay.

Adult parthenogenetic female greenhouse thrips (GHT), Heliothripshaemorrhoidalis Bouché (Thysanoptera: Thripidae) of similar age wereobtained from a colony reared on orange fruits and maintained in aninsectary at UWS Hawkesbury under conditions of 25±3° C., 65% RH and 16h D:L photoperiod

Young nymphs of citrus aphids Toxoptera citricida (Kirkaldy) (Hemiptera:Aphididae) were collected from lemon seedlings grown under glasshouseconditions at UWS Hawkesbury.

Workers of the termite Nasutitermes walkeri Hill (Isoptera: Termitidae)were collected from a laboratory culture at UWS Richmond which wasinitiated from soil, termitaria and wood on which termites were feedingwere field collected at Richmond NSW, and maintained in a darkenedcontainer under conditions of 25+2° C., 35-68% RH. Termites were fed onwood collected from near the original nest. Moistened soil from the nesttogether with paper towel were placed on top of the nest and made itpossible to maintain this culture for several months in the laboratoryat UWS Hawkesbury.

Workers of the whitefooted house ant Technomyrmex albipes (F. Smith)(Hymenoptera: Formicidae) were field collected at UWS Hawkesbury bybaiting in an empty glass jar containing sugar granules.

Pupae of housefly, Musca domestica L (Diptera: Muscidae) and differentstages of American Cockroach, Periplaneta americana L (Blattodea:Blattidae) were initially supplied by C.E.R.I.T and maintained inlaboratory culture at UWS Hawkesbury.

Tomato russet mites (TRM) Aculops lycopersici (Massee) (Acarina:Eriophyidae) were collected from infested tomato plants near Riverstone,NSW.

Mixed sex adults of the mosquito Culex quinquefaciatus (Diptera:Culicidae) were supplied by C.E.R.I.T, held at the in the Centre forHorticulture & Plant Science, University of Western Sydney, Richmond NSWand treated one day after arrival.

Workers of the honeybee Apis mellifera (Hymenoptera: Apidae) werecollected from several field hives maintained at the apiary in theCentre for Horticulture & Plant Sciences, University of Western Sydney,Richmond NSW

Adults of ash-white-flies Aleaurocanthus woglumi (Homoptera:Aleyrodidae) were field collected from an ornamental pear (Prunus sp) inthe Centre for Horticulture & Plant Sciences, University of WesternSydney, Richmond NSW.

Drug store beetle Sitodrepa panicea (Coleoptera: Anobiidae) were rearedon curry powder under laboratory conditions of 25±1° C. and 65±5% RH.

Mixed age groups of snails Helix apersa (Mollusca: Gastropoda) werecollected from infested plants in the Centre for Horticulture & PlantScience, University of Western Sydney, Richmond NSW.

Bioassays TSM

From an E. cloeziana oil extract (containing 85% tasmanone), 1.1765 gwas dissolved in 5 mL ethyl alcohol and distilled water containing 200ppm Triton X-100™ was added to prepare a 1% stock solution. From thishomogenised stock solution, further serial dilutions of 0.0125, 0.025,0.05, 0.10, and 0.12% were prepared by mixing the required amount ofstock solution in distilled water and Triton X-100 solution. Eachtreatment was conducted on 60-80 TSM, which were evenly distributed onfour French bean leaf discs (25 mm diam) contained in 90 mm diam. petridishes. The leaf discs were placed with their underside uppermost onmoist absorbent cotton wool covered with muslin netting. Water was addedto the dishes daily to prevent desiccation of the leaf discs. Five mLaliquots (unless otherwise stated) were applied to each petri dish witha Potter precision spray tower as described by Herron et al (1995). Theaverage mass of solution applied to each dish was calculated to be 3.95mg/cm². Mortality was recorded 24 h after treatment. Death wasrecognised by the absence of movement when the test organisms weremechanically stimulated. Data were analysed using SPSS for Windows™Version 7. Probit analysis was carried out for dose-mortality data andheterogeneity of regressions was determined by the Pearson chi-squaredcharacteristic.

In addition, TSM was treated with E. cloeziana oil extract incombination with paraffin oil. In particular, E. cloeziana oil extractat levels of 0.1, 0.2, 0.3, 0.4 and 0.5 g were weighed out and each wasmade up to a weight of 10.0 g with a formulated paraffin oil (BioPest®),which was then sonicated for 10 min. A 1.0% v/v of each blend wasprepared by mixing 1.0 mL with distilled water in a 100.0 mL volumetricflask. TSM were transferred to the petri dish following the samestandard method. Five-mL aliquots were applied to each petri dish andmortality was recorded 24 h after treatment. All blends of E. cloezianaoil extract with paraffin oil produced 100.0% mortality, compared withBiopest® alone which caused only 35.5±6.9 mortality. The lowestconcentration of E. cloeziana oil extract tested in this combination(i.e. 0.01%) resulted in 100% mortality in TSM, which is significantlylower than that reported earlier in this document to produce 100%mortality with E. cloeziana oil extract alone (>0.06%).

Greenhouse Thrips

The same experimental procedure used for TSM was repeated for GHT exceptthat lemon leaf discs were used instead of French beans. The requirednumber of adult thrips for each treatment was transferred with a finebrush to the underside of a lemon disc of the same diameter (6 cm) asthe base of a petri dish. The lemon disc was mounted on agar with itsadaxial side uppermost. Immediately after treatment the petri dish wascovered with perforated plastic wrap. Mortality was assessed 24 h aftertreatment.

Tomato Russet Mite

The same experimental procedure for TSM was repeated except that tomatoleaf discs were used instead of French bean.

Brown Citrus Aphid

Lemon leaf discs 2.5 cm diam. were cut from tender young leaves andmounted on moistened absorbent cotton wool in 90 mm petri dish withtheir adaxial surface uppermost. Each petri dish contained four leafdiscs. Uniform early instar nymphs were then transferred with a finebrush to the leaf discs (each containing 8-10 nymphs). A Potter towerwas used to apply 5 mL aliquots to each petri dish. A control (solventand surfactant only) was also included in the assessment. Mortality wasassessed 24 h after treatment.

Termites

Twenty uniform termite workers were transferred to 90 mm petri disheslined with the same diameter moistened filter paper (Whatman No 2). Apreliminary trial was carried out using a Potter tower to apply 5 mLaliquots of each concentration. Using this method, all termite workersdied 4 h after treatment in all concentrations, including the lowestconcentration of 0.015%. There was no mortality recorded in the blankcontrol treatment, and all workers remained alive and active for >48 hafter treatment. Identical results were obtained from theseinvestigations, whether the petri dishes were covered or uncovered afterapplication of the E. cloeziana oil extract.

Subsequent investigations further assessed efficacy of E. cloeziana oilextract by releasing termite workers on fresh dried residues. This wascarried out by uniformly distributing one mL of each concentration overthe entire surface area of a 90 mm diam filter paper. When the paper wasair dry, 20 termite workers were placed in each petri dish. One hundredpercent mortality was recorded in all four replicates even at the lowestconcentration applied (0.015% w/v=150 ppm). This suggests that the plantextract is a highly toxic contact poison to termites.

Ants

The same experimental procedure for termites was repeated for ants, withthe required number of worker ants for each treatment transferred with afine brush to the filter paper containing a fresh dried residue of theE. cloeziana oil extract. Immediately after release of the ants thepetri dish was covered with perforated plastic wrap, which enabled anyexcess vapours to escape while retaining the ants. Mortality wasassessed 4 h after treatment. In all 4 replicates 100% mortality wasobtained at concentrations as low as 0.0075% w/v ai when applied at arate of 1.0 mL to a 90 mm diam. filter paper.

Houseflies

One percent E. cloeziana oil extract was prepared using pure acetone asa diluent. From this solution, further serial dilutions were prepared byadding the required amount in acetone. Five mL aliquots of eachconcentration of E. cloeziana oil extract were dispensed into 500 mLkilner jars. The kilner jars were immediately rotated until dryness tocoat the inner surface uniformly with the E. cloeziana oil residue.After complete dryness, 30-50 pupae were transferred to a series ofclean uncovered petri dishes (45 mm diam.), one of which was placedinside each jar. The jar mouth was then covered with nylon nettingsupported by a rubber band. All adult houseflies started to emerge frompupae after 48 h and most emerged within a 3 h period. Flies were fed 5%sugar solution soaked in absorbent cotton wool. Jars were maintained inan incubator at 29° C.

Mortality was assessed at the end of the third day (i.e., approx. 72 h)after application of the E. cloeziana oil residues in the kilner jar andthe placement of pupae inside the jars. (This comprised 48 h for pupaeto emerge and 24 h exposure to E. cloeziana oil residues which were now48 h old). Flies were observed to die within a few hours afteremergence, whereas in the control they remained alive for >48 h afteremergence. The total number of adult houseflies that emerged in eachkilner jar was counted and their mortality was recorded.

American Cockroaches

Tests were conducted on 10-20 three months old nymphs (mean individualmass 0.2-0.3 g) and replicated three times. One mL of 1.0% E. cloezianaoil extract in acetone was uniformly distributed on 90 mm diam. WhatmanNo 2 filter paper. A control treatment was also carried out using 1 mLacetone only (i.e., minus E. cloeziana oil extract). After completedryness of the filter paper, the required number of cockroaches wastransferred inside the kilner jars, and were fed dry dog food. Kilnerjar necks were covered with muslin netting supported by rubber bands.Mortality was assessed 24 h after releasing the cockroaches, and deathwas recognised by the absence of movement when the test animals weremechanically stimulated.

Adult Mosquitoes

A 0.3576 g of 85% ai of E. cloeziana oil extract was dissolve in pureacetone as a diluent to give approximately 0.304% concentration stocksolution. From this solution, further serial dilutions 0.152, 0.076,0.0043 and 0.00215 were prepared by adding the required amount inacetone. Aliquots (2.5 mL) of each concentration of E. cloeziana oilextract were dispensed into 500 mL kilner jars with total internalsurface area as 286.53 cm². The kilner jars were immediately rotated tocoat the inner surface uniformly with the E. cloeziana oil extractresidue, until dry. Once completely dry, 10-25 mixed sex adult mosquitoswere released into each kilner jar by allowing the mosquitoes to flyfrom a darkened cage into the naturally lit jars. The jar mouth wassubsequently placed on 110 mm diam. filter paper onto which had beenplaced a yellow sponge soaked in 7.0% sugar solution. Treated jars werekept under laboratory temperature and humidity conditions, (viz. 24±1°C. and 65±5% RH respectively). Mortality was assessed 24 h afterreleasing the mosquitoes in the jars.

Honey Bees

Ten worker honey bees, anaesthetised with carbon dioxide, weretransferred to 90 mm diam. petri dishes lined with moistened filterpaper. Five mL aliquots were applied using a Potter Spray Tower whilethe bees were still anaesthetised. Yellow sponges soaked in 7% sugarsolution were then placed inside petri dishes for bee sustenance, andthe bees after treatment were placed in these dishes. The lids that wereperforated and covered with muslin netting were placed on the dishes,and mortality was assessed 24 hours after treatment.

Ash White Flies

For each treatment, a 2.5 cm diam. leaf disc was mounted on a moistenedWhatman #2 filter paper lining the bottom of petri dishes. Adult whiteflies were anaesthetised using carbon dioxide and 30-50 adults weretransferred onto leaf discs. Serial dilutions of 0.00546, 0.0220, 0.0894& 0.1788% concentration of E. cloeziana oil extract were prepared usingdistilled water containing 200 ppm Triton X100. Three replicates weretreated for each concentration. Five-mL aliquots were applied to eachpetri dish using a Potter Spray Tower. After treatment, petri disheswere left to dry and then covered with muslin netting. Mortalityassessment was assessed 24 h after white flies had been transferred topetri dishes.

Drug Store Beetle

One mL of each concentration of E. cloeziana oil extract in pure acetonewas uniformly dispensed on 90 mm diam. Whatman No 2 filter paper. Thelatter was left for 1 h to air dry before being used to line the lid ofa 90 mm diam. petri dish. Between 10-15 mixed adults were thentransferred into the petri dish, which was sealed with Parafilm™Mortality assessment was carried out 24 h after sealing the petri dish.

Snails

Two methods were used to assess the efficacy of E. cloeziana oil extractagainst snails. In the first method different age groups of adult snailswere dipped directly into a solution of 0.5% concentration of E.cloeziana oil extract in distilled water containing 200 ppm Triton X10.Snails were dipped for 10 seconds and thereafter immediately filtered ina sieve. The treated snails were divided in three 500 mL kilner jarscontaining French bean leaves as a food source. The control was carriedout using 0% E. cloeziana oil extract. Mortality was assessed 24 h aftertreatment.

Allowing the snails to crawl on a E. cloeziana oil extract contaminatedsurface carried out the second method. Two mL of 0.08% concentration ofE. cloeziana oil extract in pure acetone were dispensed in 500 mL kilnerjars which were rotated to uniformly cover all inner surfaces until dry.Six different age groups of snails were put into each jar along withplant material for food and covered with muslin netting held by a rubberband. Three replicates of each treatment (0.0 & 0.08% concentration)were carried out. Mortality was assessed 24 h after releasing the snailsinside the jars.

The above bioassay results are summarised in Table 8.

TABLE 8 Summary of E. cloeziana Oil Extract Efficacy Against VariousTarget Pests Method of Target Pest LD₅₀ (95% CL) LD₉₅ (95% CL)application Remarks TSM 0.07 0.14 Potter spray Knockdown effect(0.06-0.08) (0.12-0.16) tower; Aliquot observed 2 h after applied 2.5 mLtreatment TSM <0.01 E. cloeziana oil 100% mortality at extract +paraffin 0.01% oil TRM 0.02 0.04 Potter spray tower GHT 0.10 0.125Potter spray tower Aphid 0.08 0.30 Potter spray tower American 157.27μg/cm² E. cloeziana oil cockroach residues on filter nymph paper Ant<0.0075 E. cloeziana oil 100% mortality at residues on filter 0.0075%when 1 mL paper applied as residue on filter paper Termite <0.0075 E.cloeziana oil 100% mortality at residues on filter 0.0075% when 1 mLpaper applied as residue on filter paper DBM 1^(st) 0.09 0.20 5 mLPotter spray instar larva tower Housefly 69.8 μg/cm² 130.87 μg/cm² E.cloeziana oil adult residues on kilner jar wall Adult 0.00387 0.00694Residues on kilner Apparently very mosquitoes (0.00328-0.00451)(0.00596-0.01051) jar wall rapid knock down. Ash white 0.01773 0.0.03728Potter spray Assessed 4 h after fly (0.01179-0.03446) (0.02657-0.08337)tower, 2.5 mL treatment aliquot Drug store   0.03548 0.37275 SelfMortality assessed beetle contaminating by 24 h after releasing walkingon filter adults. paper Honeybees 0.12 0.40 Potter spray Knockdowneffect tower observed 2 h after Aliquot applied 5 treatment mL Snails~0.084 E. cloeziana oil Three mL acetone residues on kilner solution foreach of jar wall 3 replicates. Snails crawled on the wall on driedresidues. Snails 100% mortality Dipping method Three replicates in 0.5%

Potted Plant Investigations

The efficacy of E. cloeziana oil extract against TSM was furtherassessed under greenhouse conditions. French bean plants were grown in15 cm diam. plastic pots in an insecticide-free glasshouse maintained at27° C., RH 65% and natural light. Plants were used when they reached thetwo true-leaf stage (i.e., before trifoliate leaves appeared). One plantwas maintained in each pot (i.e., two leaves/pot). Twenty gravid TSMfemales were then transferred with a fine brush to the upper surface ofeach leaf. Mites were left to settle for 4 h before treatment, duringwhich time they usually settled on the lower leaf surfaces. E. cloezianaoil extract at a concentration of 0.07% as well as a blank control wereprepared using the same procedures described above for the laboratorybioassays. A 400 mL hand sprayer was used to apply the pesticide evenlyto all aerial surfaces of the plants, to run-off. Each treatment wasreplicated four times. The mortality was assessed 24 h after treatment.The results were recorded as mean percent mortality with standarddeviation. These results revealed that the mean percent mortality in E.cloeziana oil extract treatment and control were 92.19±6.39 and0.25±0.46, respectively.

In summary, E. cloeziana oil extract was efficacious against all peststested namely TSM, TRM, GHT, aphids, termites, houseflies, Americancockroaches, whitefooted ants adult mosquitoes, ash whitefly, drug storebeetle and snails. It was also toxic to honey bees.

Example 8 E. cloeziana Oil Extract as a Fumigant

An investigation was undertaken to determine the fumigation action of E.cloeziana oil extract against arthropods. The test organisms used weretermites, Nasutitermes walkeri. A Whatman No 2 filter paper (90 mmdiam.) was immersed in distilled water five seconds and left to drainexcess water before placing it on the bottom of a 90 mm diam. petridish, to provide moisture for termite workers during the experimentalperiod. A 1.0% E. cloeziana oil solution was prepared in pure acetone.One mL was uniformly spread on a second filter paper, which was allowedto air dry on a sheet of aluminium foil. It was then placed under thelid of the petri dishes containing the moist filter paper on their base.Twenty uniform worker termites were transferred to the moist base ofeach test petri dish, which was subsequently covered with its lidcontaining the treated filter paper, and the dishes were then sealedwith Parafilm™. A similar series of control treatments was alsoprepared, using acetone only, for comparison. Five replicates were usedin each treatment.

The termites did not move to the dry top surface and remained on thewater-moistened filter paper lining the base of the petri dishesthroughout the experimental period. Mortality was assessed 5 h aftertermite release.

The results revealed that E. cloeziana oil extract has highlysignificant fumigant effects on termites. One hundred percent mortalitywas recorded in all replicates of the E. cloeziana oil treatment whereasno mortality occurred in any of the control (acetone only) replicates.

Example 9 Efficacy of Purified β-Triketones Against TSM

The efficacy of purified β-triketones against TSM was investigated usingthe TSM bioassay described in Example 8. All β-triketones testeddemonstrated a high level of activity against TSM (see Table 9).

TABLE 9 Efficacy of β-Triketones Against TSM Sample No. wt (mg) ChemicalName LD₅₀ and 95% CL LD₉₅ and 95% CL 1 109.2 Agglomerone 0.15(0.11-021)  0.33 (0.22-1.06) 2 99.5 Grandiflorone 0.04  0.13  3 165.0Dehydroangustione 0.36 (0.33-0.41) 0.69 (0.61-0.81) 4 107.1 Angustione0.22 (0.21-0.24) 0.35 (0.31-0.40) 5 67.9 Jensenone No direct mortalityoccurred in any concentration tested (0.05-0.4%) within 24 h. However,all treated TSM were unable to move normally and continued to convulseuntil they commenced to die 72 h after treatment. There was no recovery.6 110.1 Tasmanone 0.055 0.150 7 101.8 Flavesone  0.020 (0.006-0.043)0.0876 (0.076-0.114) 8 77.0 Leptospermone 0.037 0.169 9 113.4Isoleptospermone  0.043 (0.027-0.057)  0.071 (0.058-0.109) 10 141.1Platyphyllol 0.070 0.23 

Example 10 Efficacy of Purified β-Triketones Against GHT

The efficacy of purified β-triketones against GHT was investigated usingthe GHT bioassay described in Example 8. All β-triketones testeddemonstrated a high level of activity against GHT (see Table 9).

All β-triketones except jensenone caused 100% mortality on GHT at aconcentration of 0.3% when 5 mL aliquots were applied with a Potterspray tower. The latter β-triketone did not cause direct mortalitywithin 24 h, but caused behavioural effects at all concentrationstested. Convulsion and lack of movement were consistently observed and60.0% mortality was recorded 72 h after application in the 0.4%treatment.

Examples 11

Ready-to-use miticide spray-I Ingredient Parts E. cloeziana extract 0.1Emulsifier: e.g. t-octylphenoxypolyethoxyethanol, 1.0polyoxyethylenesorbitan, organosilicate Solvent: e.g. ethyl alcohol,isopropyl alcohol etc 5 Tannic acid 1.0 Carrier e.g. water 92.9

Examples 12

Concentrated natural emulsifiable concentrate spray (4.4%)-I IngredientParts E. cloeziana extract 4.4 Pyrethrins 7.4 Emulsifier: e.g.t-octylphenoxypolyethoxyethanol, 14.7 polyoxyethylenesorbitan,organosilicate Solvent: e.g. ethyl alcohol, isopropyl alcohol etc 73.5

Example 12

Concentrated emulsifiable concentrate spray (44%) Ingredient Parts E.cloeziana extract 22.0 Platyphyllol (natural or synthetic) 22.0Emulsifier: e.g. t-octylphenoxypolyethoxyethanol, 34.0polyoxyethylenesorbitan, organosilicate Solvent: e.g. ethyl alcohol,isopropyl alcohol etc 22

Example 13

Natural ready to use insecticide spray Ingredient Parts E. cloezianaextract 0.3 Lavender oil 1.0 Emulsifier: e.g.t-octylphenoxypolyethoxyethanol, 1.0 polyoxyethylenesorbitan,organosilicate Solvent e.g. ethyl alcohol, isopropyl alcohol etc 40Carrier: Water 57.7

Example 14

Oil-based natural spray concentrate Ingredient Parts E. cloezianaextract 10.0 Petroleum oil 89.0 Emulsifier: e.g.t-octylphenoxypolyethoxyethanol, 1.0 polyoxyethylenesorbitan,organosilicate

Example 15

Concentrated emulsifiable concentrate spray (10%) Ingredient Parts E.cloeziana extract 10.0 Permethrin 10.0 Piperonyl butoxide 28.0Emulsifier: e.g. t-octylphenoxypolyethoxyethanol, 31.0polyoxyethylenesorbitan, organosilicate Solvent e.g. ethyl alcohol,isopropyl alcohol etc 21

Example 16

Molluscidal dust Ingredient Parts E. cloeziana extract 2 Anti-cakingagent (e.g. silica gel) 2 Emulsifier: e.g.t-octylphenoxypolyethoxyethanol, 3 polyoxyethylenesorbitan,organosilicate Inert carrier (talc, kaolin, diatomaceous earth) 93

Example 17

Aerosol insecticidal and acaricidal spray Ingredient Parts E. cloezianaextract 1.0 Piperonyl butoxide 0.9 Propellent hydrocarbon 98.1

Example 18

Repellent Ingredient Parts E. cloeziana extract 19.5 Citronella oil 29.1Phthalic acid dibutyl ester 29.1 N-octyl bicycloheptene dicarboxamide22.3

Examples 19

Ready-to-use miticide spray-II Ingredient Parts Melaleuca cajeputiextract 0.2 Emulsifier: e.g. t-octylphenoxypolyethoxyethanol, 1.0polyoxyethylenesorbitan, organosilicate Solvent: e.g. ethyl alcohol,isopropyl alcohol etc 5.0 Tannic acid 1.0 Carrier e.g. water 92.8

Examples 20

Concentrated natural emulsifiable concentrate spray (4.4%)-II IngredientParts Melaleuca cajeputi extract 8.8 Pyrethrins 7.4 Emulsifier: e.g.t-octylphenoxypolyethoxyethanol, 14.7 polyoxyethylenesorbitan,organosilicate Solvent: e.g. ethyl alcohol, isopropyl alcohol etc 69.1

Examples 21

Ready-to-use miticide spray-III Ingredient Parts 99% Tasmanone 0.09Emulsifier: e.g. t-octylphenoxypolyethoxyethanol, 1.0polyoxyethylenesorbitan, organosilicate Solvent: e.g. ethyl alcohol,isopropyl alcohol etc 5 Tannic acid 1.0 Carrier e.g. water 92.91

Examples 22

Concentrated natural emulsifiable concentrate spray (4.4%)-IIIIngredient Parts 99% Tasmanone 4.0 Pyrethrins 7.4 Emulsifier: e.g.t-octylphenoxypolyethoxyethanol, 14.7 polyoxyethylenesorbitan,organosilicate Solvent: e.g. ethyl alcohol, isopropyl alcohol etc 73.9

Examples 23

Ready-to-use miticide spray-IV Ingredient Parts 99% Platyphyllol 0.09Emulsifier: e.g. t-octylphenoxypolyethoxyethanol, 1.0polyoxyethylenesorbitan, organosilicate Solvent: e.g. ethyl alcohol,isopropyl alcohol etc 5 Tannic acid 1.0 Carrier e.g. water 92.91

Examples 24

Concentrated natural emulsifiable concentrate spray (4.4%)-IV IngredientParts 99% Platyphyllol 4.0 Pyrethrins 7.4 Emulsifier: e.g.t-octylphenoxypolyethoxyethanol, 14.7 polyoxyethylenesorbitan,organosilicate Solvent: e.g. ethyl alcohol, isopropyl alcohol etc 73.9

Example 25 Intra-Specific Crosses for Imparting Pest Resistance to aPest Susceptible Plant

Controlled and wild pollination within Eucalyptus and other importantcommercial Myrtaceae are thoroughly addressed in the CRC for SustainableProduction Forestry Symposium on Hybrid Breeding and Genetics—ControlledPollination of Eucalypts on 12th April, 2000 and published as theproceedings of that symposium. Genetic Pollution from Farm Forestry(Potts et al., 2001) deals more specifically with intra and interspecies crosses occurring within the Myrtaceae.

Using the protocols described in the above publications, anintra-specific Eucalyptus cross breeding technique will employ thefollowing protocol. This begins with the selection of supremeindividuals as parent stock. Within a selected parent stock havingsuperior pest resistant characteristics (e.g., E. cloeziana) male andfemale trees are identified for cross-pollination experiments. Pollen isharvested from the male trees and either stored or directly transferredto the female trees if flowering is synchronous. Emasculation isundertaken to preclude extraneous pollination occurring and flowers areoften bagged as a further precaution. Seed set and subsequent embryodevelopment proceeds over the ensuing 12-24 months and F1 seeds arecollected at full maturation.

Seeds are then germinated to produce seedlings that are subjected todetailed analysis to assess the transfer of traits from parent toprogeny. If the F1 progeny show a desirable mix of phenotypic traitsthese progeny can be used to vegetatively propagate the new variety. Ifthe F1 progeny show some improvement in the selected phenotypic traits,but further improvement is required selected F1 progeny can beback-crossed either within the F1 progeny or with one of the parenttrees to produce an F2 progeny using the methodology outlined above.This iterative process can be continued ad infinitum until the desiredcharacteristics are achieved.

Example 26 Inter-Specific Crosses for Imparting Pest Resistance to aPest Susceptible Plant

Inter-species hybridisation in the wild, which is a common phenomenonwithin the subgenera of the Myrtaceae, have been recorded at almost 40%in the Eucalypts, 33% in Angophora 19% in Corymbia and 19% inSymphomyrtus. Hybridisation between the major subgenera may also occur(Potts et al., 2001). For example, Eucalyptus camaldulensis displays 14natural hybrid crosses including E. camaldulensis×E. robusta, E.camaldulensis×E. alba, E. camaldulensis×E. cladocalyx, E.camaldulensis×E. bigalaterita, E. camaldulensis×E. tereticornis, E.camaldulensis×E. blakelyi, E. camaldulensis×E. dwyeri, E.camaldulensis×E. rudis, E. camaldulensis×E. ovata, E. camaldulensis×E.bridgesiana, E. camaldulensis×E. viminalis, E. camaldulensis×E.largiflorens, E. camaldulensis×E. melliodora, E. camaldulensis×E.leucoxylon; 15 manipulated hybrids including E. camaldulensis×E.diversicolor, E. camaldulensis×E. grandis, E. camaldulensis×E.botryoides, E. camaldulensis×E. cladocalyx, E. camaldulensis×E.tereticornis, E. camaldulensis×E. blakelyi, E. camaldulensis×E.urophylla, E. camaldulensis×E. macarthurii, E. camaldulensis×E. exerta,E. camaldulensis×E. maidenii, E. camaldulensis×E. viminalis, E.camaldulensis×E. globulus, E. camaldulensis×E. gunnii, E.camaldulensis×E. laevopinea, E. camaldulensis×E. fastigata. In anotherexample Eucalyptus globulus displays 15 natural hybrids including E.globulus×E. barberi, E. globulus×E. brookeriana, E. globulus×E. ovata,E. globulus×E. kitsoniana, E. globulus×E. goniocalyx, E. globulus×E.nortonii, E. globulus×E. cypellocarpa, E. globulus×E. pseudoglobulus, E.globulus×E. bicostata, E. globulus×E. johnstonii, E. globulus×E.viminalis, E. globulus×E. cordata, E. globulus×E. rubida, E. globulus×E.urnigera, E. globulus×E. perriniana and 13 successful manipulatedhybrids including E. globulus×E. urophylla, E. globulus×E. grandis, E.globulus×E. robusta, E. globulus×E. pellita, E. globulus×E. longifolia,E. globulus×E. loxophloeba, E. globulus×E. camaldulensis, E. globulus×E.dunnii, E. globulus×E. nitens, E. globulus×E. maidenii, E. globulus×E.bicostata, E. globulus×E. viminalis, E. globulus×E. gunnii. In anotherexample Eucalyptus grandis displays 4 natural hybrids including E.grandis×E. urophylla, E. grandis×E. robusta E. grandis×E. pellita E.grandis×E. terreticornis and 14 manipulated hybrids including E.grandis×E. urophylla E. grandis×E. botryoides E. grandis×E. pellita E.grandis×E. alba E. grandis×E. terreticornis E. grandis×E. camaldulensisE. grandis×E. dunnii E. nitens×E. maidenii E. grandis×E. globulus E.grandis×E. gunnii E. grandis×E. pulverulenta E. grandis×E. leucoxylon,E. grandis×E. resinifera. In another example the Corymbiahenryi/variegata/maculata/citriodora complex displays 14 natural hybridsincluding C. citriodora×C. catenaria, C. citriodora×C. variegata, C.citriodora×C. maculata, C. maculata×C. gummifrea, C. maculata×C.intermedia, C. maculata×C. citriodora, C. maculata×C. variegata, C.variegata×C. bloxsomeri, C. variegata×C. watsoniana, C. variegata×C.citriodora, C. variegata×C. maculata, C. Henryi×C. torelliana, C.henryi×C. variegata and onemanipulated hybrid C. torelliana×c. In onefurther example Eucalyptus cloeziana displays only 1 natural hybrid E.cloeziana×E. acmenoides.

The disclosure of every patent, patent application, and publicationcited herein is hereby incorporated herein by reference in its entirety.

The citation of any reference herein should not be construed as anadmission that such reference is available as “Prior Art” to the instantapplication

Throughout the specification the aim has been to describe the preferredembodiments of the invention without limiting the invention to any oneembodiment or specific collection of features. Those of skill in the artwill therefore appreciate that, in light of the instant disclosure,various modifications and changes can be made in the particularembodiments exemplified without departing from the scope of the presentinvention. All such modifications and changes are intended to beincluded within the scope of the appended claims. All figures, tables,and appendices, as well as publications, patents, and patentapplications, cited herein are hereby incorporated by reference in theirentirety for all purposes.

I. BIBLIOGRAPHY

-   Bignall, C. M., Dunlop, P. J., Brophy, J. J. and Fookes, C. J. R.    (1997). Volatile Leaf Oils of some South-western and Southern    Australian Species of the Genus Eucalyptus (Series I). Part XIV.    Subgenus Monocalyptus. Flav Frag. Journal, 12, 177-183.-   Boland, D. and J. Brophy (1993). Essential Oils of the Eucalyptus    and Related Genera: Search for Chemical Trends. Bioactive Volatile    Compounds from Plants. R. Teranishi, R. G. Buttery and H. Sugisawa.    Washington D. C., American Chemical Society. 525: 72-87.-   Brophy, J. J. and D. J. Boland (1990). “Leaf Essential Oil of Two    Chemotypes of Eucalyptus cloeziana F. Muell.” Journal of Essential    Oil Research 2 (March/April): 87-90.-   Brophy, J. J., R. J. Goldsack, et al. (1995). “Leaf Oils of the    Genus Backhousia (Myrtaceae).” Journal of Essential Oil Research 7    (May/June): 237-254.-   CRC for Sustainable Production Forestry. Symposium on Hybrid    Breeding and Genetics—Controlled Pollination of Eucalypts, Noosa    Australia, 12th April, 2000-   Hellyer, R. (1968). “The Occurrence of 3-Triketones in the    Steam-Volatile Oils of some Myrtaceous Australian Plants.” Aust. J.    Chem. 21(11): 2825-2828.-   Herron G A, Beatie G A C, Parkes R A & Barchia I. 1995. Potter spray    tower bioassay of selected citrus pests to petroleum spray oil.    Journal of Australian Entomological Society 34: 225-263.-   Potts, B M, Barbour, R C and Hingston, A H, 2001. Genetic Pollution    from Farm Forestry. Rural Industries Research and Development    Corporation Publication (No 01/114).-   Southwell, I. A. and J. J. Brophy (2000). “Essential oil isolates    from the Australian Flora. Part 3.” Journal of Essential Oil    Research 12: 267-278.

What is claimed is:
 1. A method for controlling insects, arachnids,molluscs, protozoa and helminths, said method comprising exposing theinsects, arachnids, molluscs, protozoa and helminths to apest-controlling effective amount of the compound flavesone(1-isobutyroyl-3,3,5,5-tetramethylcyclohexan-2,4,6-trione).
 2. Themethod of claim 1, wherein the compound is obtainable from a volatileoil-bearing organism.
 3. The method of claim 2, wherein the volatileoil-bearing organism is selected from volatile oil-bearing plants. 4.The method of claim 2, wherein the volatile oil-bearing organism isselected from genera of the Myrtaceae family.
 5. The method of claim 2,wherein the volatile oil-bearing organism belongs to a genus selectedfrom Angophora, Austromyrtus, Backhousia, Baeckea, Callistemon,Corymbia, Darwinia, Eucalyptus, Kunzea, Leptospermum, Melaleuca,Syzygium and Xanthostemon
 6. The method of claim 1, wherein the pestthat is controlled is selected from insects, arachnids and molluscs. 7.The method of claim 1, wherein the helminth is a nematode.
 8. The methodof claim 1, wherein the compound is used in the form of apest-controlling composition which comprises from about 0.00005% toabout 90% by weight of said compound.